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Cy5 total protein normalization in Western blot analysis
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Åsa Hagner-McWhirter ⇑, Ylva Laurin, Anita Larsson, Erik Bjerneld, Ola Rönn
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GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden
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a r t i c l e
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Article history: Received 12 March 2015 Received in revised form 10 June 2015 Accepted 11 June 2015 Available online xxxx Keywords: Western blotting Normalization Loading control CyDye Prelabeling Housekeeping protein
a b s t r a c t Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0 lg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence. Ó 2015 Elsevier Inc. All rights reserved.
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Western blotting is used for qualitative and quantitative analysis of proteins. Qualitative analysis may confirm the identity, presence, or absence of a protein or provide a rough estimate of the amounts of protein. Uneven sample loading is common owing to errors in protein quantitation, cell number estimation, or pipetting. Therefore, to quantitatively compare the levels of specific proteins between samples in Western blots, it is common to relate the target signal to a presumably constitutively expressed endogenous or a so-called housekeeping protein such as glyceraldehyde-3phosphate dehydrogenase (GAPDH)1 or structural proteins such as b-tubulin and b-actin. The signal of the housekeeping protein is expected to be proportional to total protein and hence the cell number loaded in each well. However, this method has many experimental pitfalls, such as cell- or tissue-dependent linear response range of the sample, suboptimal dilution, and/or poor quality of the antibodies, which may weaken the proportionality of signal-to-sample loading. In addition, the reliability of using housekeeping proteins as loading controls is under scrutiny because ⇑ Corresponding author. Fax: +46 18 6121844. E-mail address: [email protected] (Å. Hagner-McWhirter). 1 Abbreviations used: BSA, bovine serum albumin; PVDF, polyvinylidene difluoride; CHO, Chinese hamster ovary; PP2A, protein phosphatase type 2A; EGF, epidermal g r o w t h f a c t o r ; E R K , e x t r a c e l l u l a r s ig n a l - r e g u l a te d k i n a s e ; G A P D H , glyceraldehyde-3-phosphate dehydrogenase; NHS, N-hydroxysuccinimide; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; DTT, dithiothreitol; RuBPS, ruthenium(II)–tris(bathophenanthroline disulfonate).
culture conditions or treatments may affect the expression level of housekeeping proteins [1–3]. It is important to choose an appropriate method to detect the protein signal and it is critical to qualify target and control signals to be used for normalization. Housekeeping proteins are widely used but not always reliable as loading controls and an increased interest in using total protein instead has been seen in recent publications [1,4–7]. A fluorescence-based detection system produces stable signals, high sensitivity, broad dynamic range, and the ability to normalize by detecting several targets simultaneously by multiplexing on the same blot. Chemiluminescence detection requires more stringent experimental control, and the lack of multiplexing capability necessitates stripping and reprobing procedures that may increase the risk of experimental variation. Using the total protein signal from a sample lane for normalization has been investigated previously and shown to be the most reliable loading control. The total protein signal was obtained by poststaining the membrane with Ponceau S [7], RuBPS [4], and Coomassie [5] stains or detected with ultraviolet radiation [6]. We present a reliable and easy normalization method based on Cy5-prelabeled total protein. The labeling reaction is based on NHS chemistry with a ready-to-use Cy5 dye reagent [8]. The Cy5-labeled proteins are then transferred to the membrane followed by probing with primary antibody against target protein and Cy3-labeled secondary antibodies in a multiplex experiment on the same blot. The Cy5 total protein signal for each lane on
http://dx.doi.org/10.1016/j.ab.2015.06.017 0003-2697/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Å. Hagner-McWhirter et al., Cy5 total protein normalization in Western blot analysis, Anal. Biochem. (2015), http:// dx.doi.org/10.1016/j.ab.2015.06.017
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the membrane is used as a control signal for normalization. We provide comparative performance evaluations of total protein normalization and endogenous protein normalization using housekeeping proteins, in addition to quantitative Western blot applications in which total protein was used for normalization.
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Materials and methods
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Total protein concentration was determined using a Coomassie Plus protein assay kit (Thermo Scientific). The Cy5 prelabeling reaction volume was adjusted to 19 ll with original lysis buffer for all the samples, and 1 ll of Amersham WB Cy5 dye reagent (approximately 250 pmol Cy5–NHS/ll; GE Healthcare), which had been freshly diluted 1:10 or 1:20 with ultrapure water just prior to labeling, was added to start the labeling reaction. The samples were mixed and incubated for 30 min at room temperature or on ice. The reactions were stopped by the addition of 20 ll of Amersham WB loading buffer (50 mM Tris–Cl, 0.25% (w/v) orange G, 4% (w/v) SDS, 0.5 mM lysine; GE Healthcare) containing 40 mM DTT. The reaction volumes were sometimes scaled up two- or fivefold to a final reaction volume of 40 or 100 ll. The samples were heated for 3–5 min at 95 °C and 20 ll of each sample was applied to the wells of an Amersham WB gel card 14, 8–18% (GE Healthcare). Amersham WB molecular weight markers (GE Healthcare) were diluted 1:60 or 1:80 in diluted loading buffer with 40 mM DTT (1 part loading buffer plus 1 part ultrapure water). We performed electrophoresis in an Amersham WB system (GE Healthcare), at 600 V for 45–50 min. The Cy5-prelabeled and unlabeled proteins were transferred to an Amersham PVDF card (GE Healthcare) using a tank transfer protocol and a transfer buffer (192 mM glycine, 25 mM Tris) containing 20% (v/v) ethanol at 100 V for 30 min. The Amersham WB system default probing protocol was used. The membranes were blocked in 3% (w/v) BSA in PBS-T or TBS-T (containing 0.1% (v/v) Tween 20) for 10 min, and a PBS-T or TBS-T buffer was used for the dilution and wash steps. PBS or TBS buffer was used for the final wash. The control and target primary antibodies were mixed in a total probing volume of 10 ml and incubated for 1–3 h followed by secondary antibody incubation for 30 min using Amersham WB Cy3- or Cy5-labeled secondary antibodies (GE Healthcare). The membranes were dried for 10 min in the system before scanning in the Cy3 and Cy5 channels of the Amersham WB system using automatic or manual settings. We used the Amersham WB evaluation software with built-in normalization to determine target, control signals, and normalized ratio. Coefficients of variation (CVs) were calculated using Microsoft Excel software. The background was subtracted from the target protein bands and Cy5-labeled total protein with rolling ball sizes of 400 and 1000 (default background subtractions), respectively.
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Linearity of Cy5 total protein labeling reaction
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Chinese hamster ovary (CHO) cells expressing monoclonal antibodies (suspension cell culture) were grown in a 50-L WAVE bag (working volume 25 L) on a WAVE bioreactor system (GE Healthcare). Cells were grown in ActiCHO medium (GE Healthcare) and from day 3 fed with ActiCHO Feed-A and ActiCHO Feed-B (GE Healthcare) daily. From day 6, the glucose concentration was measured and glucose administered to the culture up to 6 g/L daily. The cells were harvested by centrifugation at 3000g for 20 min at room temperature. The cell pellet was washed twice by resuspending the pellet in a fourfold volume of PBS buffer, followed by centrifugation (as above), and discarding the
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supernatant. The washed cell pellet was lysed for 1.5 h on ice with occasional mixing in 1 ml of Mammalian Protein Extraction Buffer (GE Healthcare) containing 10 ll/ml Protease Inhibitor Mix (GE Healthcare) per approximately 1 ml of cell pellet. Cell debris was removed by centrifugation at 16,000g for 30 min at +4 °C. The cell lysate supernatant was transferred to a new tube. The linearity of the labeling reaction was analyzed by incubating various amounts of total protein with the Cy5 dye reagent. Duplicate tubes containing 10, 20, 40, 80, 160, 240, and 300 lg of cell lysate per 20 ll were prepared and labeled with Cy5 dye reagent (which had been diluted 1:10) in equal final reaction volumes of 100 ll of lysis buffer. The samples described above were incubated at room temperature (approximately +22 °C) and at +4 °C (on ice) in parallel. The reactions were stopped by the addition of an equal volume (100 ll) of loading buffer. The samples were diluted 1:5 in diluted loading buffer (1 part loading buffer and 1 part ultrapure water) and 20-ll samples containing 1, 2, 4, 8, 16, 24, or 30 lg of CHO cell lysate were applied to the wells of the gel. The gels were scanned after electrophoresis, and the membranes were scanned after transfer in the Cy5 channel. Linearity for lower amounts of total proteins was investigated in a separate experiment (as above) with reactions containing 5, 10, 20, and 40 lg of CHO cell lysate. Samples (20 ll) without dilution were applied to the wells of the gels and scanned after electrophoresis in the Cy5 channel. The Cy5 total protein signal from each lane was plotted against the amount of protein.
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Detection of ERK1/2 in various amounts of cell lysate
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CHO cells were lysed as described above and a dilution series with incremental steps of 5 (from 5 to 40 lg per 20-ll reaction volume) was prepared in duplicate. The volume was adjusted to 95 ll with lysis buffer for all protein amounts, and 5 ll of Cy5 dye reagent (which had been diluted 1:20 in ultrapure water) was added to start the labeling reaction. The duplicate reactions were stopped by the addition of an equal volume (100 ll) of loading buffer and 20 ll of each sample, containing 2.5 to 20.0 lg of cell lysate, was loaded per well. The unlabeled samples were prepared in parallel but 5 ll of ultrapure water was added instead of the diluted Cy5 dye reagent and 20 ll of each sample was loaded per well. ERK1/2 was targeted by rabbit anti-mitogen-activated protein kinase (MAPK) primary antibody (Sigma–Aldrich) that had been diluted 1:5000 and Cy3-labeled (anti-rabbit IgG) secondary antibody, which had been diluted 1:2500. Endogenous control proteins were targeted in the unlabeled samples by mouse anti-tubulin and mouse anti-actin primary antibodies (both of which had been diluted 1:5000) and mouse anti-GAPDH primary antibody, which had been diluted 1:2500 (Sigma–Aldrich). After it was verified that there was no cross-reaction, the three mouse primary antibodies were mixed and incubated for 1.5 h at room temperature. This was followed by incubation with Cy5-labeled (anti-mouse IgG) secondary antibody, which had been diluted 1:2500, for 1 h at room temperature. The membranes were dried and scanned in the Cy3 and Cy5 channels. The ERK1/2 signals were normalized against Cy5 total protein or Cy5 tubulin, actin, or GAPDH endogenous protein signals.
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Reproducibility of Cy5 total protein normalization
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CHO cell lysates were prepared as described above and three different operators performed Cy5 prelabeling of 8, 24, and 40 lg in quadruplicate reactions using Cy5 dye reagent diluted 1:10. The reactions were stopped with an equal volume of loading buffer and heated as described above. The samples were subjected to
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Western blotting using three different membranes per operator. Tubulin was targeted with mouse anti-tubulin (Sigma–Aldrich), which had been diluted 1:1000, and Cy3-labeled (anti-mouse IgG) secondary antibody, which had been diluted 1:2500. The dried membranes were scanned in the Cy3 and Cy5 channels. The Cy3 tubulin signals were normalized using Cy5 total signal and the CV of the normalized ratio (Cy3/Cy5) between 4 and 20 lg was calculated for each operator and membrane.
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Detection of ERK1/2 in Cy5-prelabeled and unlabeled sample
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CHO cells were lysed as described above and samples containing 20 or 40 lg per 20-ll volume were prepared in duplicate. Cy5 dye reagent (diluted 1:10) was used to prelabel one sample set in a final volume reaction volume of 100 ll lysis buffer. An equal volume (100 ll) of loading buffer containing 40 mM DTT was added to both the Cy5-prelabeled (to stop the reaction) and the unlabeled samples; 20-ll samples containing 10 or 20 lg of CHO cell lysate were applied in triplicate to the same SDS–PAGE gel. Rabbit anti-MAPK primary antibody (Sigma–Aldrich) was diluted 1:5000 and Cy3-labeled (anti-rabbit IgG) secondary antibody was diluted 1:2500 and used to target ERK1/2. The dried membranes were scanned in the Cy3 and Cy5 channels. The target Cy3 signal intensity of ERK1/2 was compared for Cy5-prelabeled and unlabeled samples.
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Detection of actin and phospho-protein phosphatase type 2A (PP2A) in epidermal growth factor (EGF)-stimulated A431 cells
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Cell lysates (2.5 lg/ll) of A431 (human epidermal carcinoma cell line) cells stimulated with EGF were purchased from Santa Cruz Biotechnology. Lysates of unstimulated A431 (control) cells were mixed with increasing amounts (0, 25, 50, 75, and 100%) of EGF-stimulated cell lysates and prelabeled with diluted (1:10) Cy5 dye reagent. Duplicate samples containing 15 lg (actin detection) or 20 lg (PP2A detection) of the cell lysate were loaded onto the SDS–PAGE gel. One blot was probed for the detection of actin with mouse anti-actin primary antibody (Sigma–Aldrich) diluted 1:5000 and Cy3-labeled antibody (anti-mouse IgG) diluted 1:2500. Another blot was probed for the detection of PP2A using rabbit anti-PP2A primary antibody (C subunit (52F8); Cell Signaling), which had been diluted 1:750, and Cy3-labeled (anti-rabbit IgG) secondary antibody, which had been diluted 1:2500. The dried membranes were scanned in the Cy3 and Cy5 channels. The actin and PP2A signals were normalized against Cy5 total protein on the two separate blots.
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Detection of Smad2/3 in NMuMG cells
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NMuMG cells (mouse mammary epithelial cell line) were grown in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 0.1% insulin. The cells were starved overnight (16 h), followed by treatment with 1 ng/ml tumor growth factor-b (TGF-b) for 0, 15, 30, or 60 min or 6 h. Untreated cells (control) were grown without starvation for 24 h. The cells were washed with PBS and detached with a rubber policeman in lysis buffer containing 1% Triton X-100. The NMuMG cell lysates were prelabeled with diluted (1:10) Cy5 dye reagent. Samples containing 20 lg of the cell lysate were subjected to Western blot analysis and probed as described above with the exception that the primary antibody was incubated for 3 h instead of 1 h. TBS-T or TBS buffer was used for the dilution and wash steps. Smad2/3 was detected using mouse anti-Smad2/3 (C-8; Santa Cruz Biotechnology) diluted 1:250 and Cy3-labeled (anti-mouse IgG) secondary antibody, which had been diluted
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1:2500. The membrane was dried and scanned in the Cy3 and Cy5 channels. The Smad2/3 signals were normalized against Cy5 total protein.
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Results
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Linearity of Cy5 prelabeling of CHO cell lysates
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The signal response of labeled total protein was tested for a broad range of sample concentrations at both room temperature and +4 °C (Fig. 1A and B). The Cy5 prelabeling reaction was linear up to 80 lg per reaction (reaction volume 20 ll, corresponding to 4 lg total protein/ll) for reactions performed at room temperature and linear across the whole tested range up to 300 lg per reaction (corresponding to 15 lg total protein/ll) when the reaction was performed at +4 °C (Fig. 1C). The linearity at lower amounts of total protein was confirmed at room temperature and +4 °C (Fig. 1D). The variation in signal intensity between replicate reactions was low (Fig. 1D). The results in Fig. 1 are based on analysis in the gel, and similar results were obtained by analysis of the signals after transfer to the membrane (data not shown).
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Actin versus total protein as loading control in EGF-stimulated cells
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The same amounts of Cy5-prelabeled A431 cell lysate mixtures with increasing concentrations of EGF-stimulated lysate were subjected to Western blotting targeting the housekeeping protein actin with anti-actin primary antibodies and Cy3-labeled secondary antibodies. The Cy3 actin signals increased (Fig. 2A), but the Cy5 total protein signal was unaffected, indicating similar loading of the samples (Fig. 2B). Normalization to Cy5 total protein reveals that actin levels increased dramatically after EGF treatment (Fig. 2C). Actin was regulated by EGF treatment and thus inadequate as a loading control for this cell type and treatment, whereas the more reliable Cy5 total signal would accurately reflect the sample load.
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Quantitation of ERK1/2—comparing normalization performance using endogenous control proteins or total protein
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To compare quantitation of the target over a wide loading range, a dilution series of the same CHO cell lysate was used. To measure the degree of normalization efficiency, we calculated the CV% of the normalized ratio across the loading range. The ERK1/2 target signals from CHO cell lysates produced a proportional response to sample load, and variation between the duplicate samples was very low for all blots (Figs. 3 and 4). The ERK1/2 signals were normalized using either Cy5 total signal within the whole lane (Fig. 3) or one of the endogenously expressed loading control proteins actin, tubulin, or GAPDH (Fig. 4). The Cy5 total protein control signals showed low variation between duplicate labeling reactions and the total signal was proportional to sample load (Fig. 3B). A sample load of 20 lg gave a lower Cy5 total signal, but the same was observed for the Cy3– ERK1/2 antibody-mediated signal, suggesting that less sample was loaded in these wells and the reduction was not related to the Cy5 prelabeling. Normalization of ERK1/2 signals using Cy5 total protein resulted in similar target/control ratios with a CV of 7% over the whole sample loading range (Fig. 3C and Table 1). Normalization of ERK1/2 signals using tubulin, actin, or GAPDH instead resulted in ratios with a CV of 18, 13, and 54%, respectively (Fig. 4 and Table 1). Using tubulin or actin as control resulted in a higher variation of normalized values across the whole range with a visible difference in ratio between high and low sample amounts (Fig. 4A and B). GAPDH showed very poor performance since the
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Cy5 total protein Integrated intensity (volume x 10-3)
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Fig.1. Linearity of Cy5 total protein prelabeling. Duplicate Cy5 prelabeling reactions containing 10, 20, 40, 80, 160, 240, and 300 lg CHO cell lysate per reaction as indicated were incubated at (A) room temperature (RT) or at (B) +4 °C for 30 min, and fivefold-diluted samples were subjected to electrophoresis and scanning using the Amersham WB system. (C) The Cy5 total signal within the whole lane from the gels in (A) and (B) was plotted against the amount of protein in the reaction for room temperature (filled circles) and +4 °C (open circles) incubation. (D) Triplicate Cy5 prelabeling reactions containing 5, 10, 20, or 40 lg CHO cell lysate per reaction were incubated at room temperature (filled circles) or at +4 °C (open circles) for 30 min and the Cy5 total signal within the whole lane was plotted against the amount of protein in the reaction.
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Normalized ratio Cy3/Cy5
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Cy5 total protein Integrated intensity (volume x 10-3)
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Sample number
Fig.2. Activation of actin in response to EGF treatment. A431 cell lysates from control and EGF-treated cells were mixed with increasing amounts of treated lysate (0, 25, 50, 75, and 100% treated lysate), prelabeled with Cy5, and loaded in duplicates as indicated. After transfer, the membranes were probed for b-actin using anti-actin primary antibody and Cy3-labeled secondary antibody. (A) Cy3 b-actin signals, (B) Cy5 total protein, and (C) normalized Cy3/Cy5 ratio obtained after image analysis are presented in the bar graphs. 326 327 328 329
specific signal did not increase much with increasing sample load and showed almost no normalization effect, resulting in a large variation in normalized ratio over the sample range (Fig. 4C and Table 1).
Reproducibility of Cy5 total protein normalization
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Using Cy5 total protein normalization, three different operators performed Western blotting targeting tubulin using primary
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Fig.3. Normalization of ERK1/2 signals using total protein normalization. Various amounts of the same CHO cell lysate were Cy5-prelabeled in duplicate reactions. Samples containing 2.5, 5, 10, 15, and 20 lg total protein were applied to the gel as indicated. The prelabeled samples were subjected to Western blotting and the membrane was probed with anti-ERK1/2 primary antibody and Cy3-labeled secondary antibody. (A) Cy3 ERK signal, (B) Cy5 total signal within each lane, and (C) the normalized ratio of Cy3/ Cy5 obtained after image analysis are presented in the bar graphs.
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antibody and Cy3-labeled secondary antibody. For the whole sample loading range (4–20 lg) in quadruplicate reactions and all three operators and triplicate membranes, the normalized ratio had an average CV of 8% (Table 2), showing that the normalization method was very reproducible.
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Effect of Cy5 prelabeling on target signal intensity
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To investigate whether Cy5 prelabeling adversely affected antibody binding to its target, we compared ERK1/2 signals from unlabeled and Cy5-prelabeled CHO cell lysate that had been applied to the same SDS–PAGE gel. Half of the gel was loaded with Cy5-prelabeled cell lysate and the other half with unlabeled samples. The Western blot membrane was incubated with the same antibody solutions and imaged with the same settings. Triplicate samples of 10 and 20 lg of CHO cell lysate were compared under identical conditions. No significant differences in signal intensities were seen, showing that Cy5 prelabeling had no effect on the detection of ERK1/2 (Fig. 5). Using this labeling protocol, the proportion of Cy5-labeled proteins is below 5% (data not shown), leaving the majority unlabeled. This suggests that antibody binding is not likely to be affected by Cy5 prelabeling of samples.
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Analysis of PPA2 and Smad2/3 levels using Cy5 total protein normalization To demonstrate the accuracy of analysis using total protein normalization we studied the effect of EGF treatment on the expression levels of PP2A in A431 cell lysates. Cy5-prelabeled cell lysate samples with various proportions of EGF-stimulated cell lysate were subjected to Western blotting. The PP2A signals were increased with EGF treatment (Fig. 6A), and the Cy5 total protein signals were similar, indicating low variation in sample load (Fig. 6B). When the PP2A signals were normalized using Cy5 total protein as a control, the results showed that EGF stimulation caused an approximately 50% upregulation of PP2A (Fig. 6B). In
another experiment, regulation of Smad2 and Smad3 by TGF-b treatment for various lengths of time was analyzed by Western blotting using Cy5 total protein normalization. The signal intensities of Smad2 without normalization showed a downregulation (Fig. 7A). Fig. 7B shows the sample load of the various samples, and when Smad2 was normalized to Cy5 total protein a clear effect was confirmed as a result of TGF-b treatment, showing an approximately 50% downregulation at 30 and 60 min (Fig. 7C). A small difference in Smad2 level between 30 and 60 min could also be seen. Smad3 was not changed as a result of TGF-b treatment incubation time (data not shown). The control sample (0 h incubation) had a different protein composition compared to the other samples, with a high expression of proteins with molecular weights ranging between 55 and 65 kDa (Fig. 7B).
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Discussion
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The Western blotting work flow contains many steps that potentially can affect the data quality, and one of the most important factors is the normalization [8]. Normalization of target signals using housekeeping proteins or total protein signals is critical to minimize the adverse effects of loading variability in quantitative Western blotting. The sources of loading variability include errors in protein quantitation of different samples or the estimation of sample amounts (e.g., one culture dish per sample). Single endogenous loading controls are often used routinely without confirming their normalization performance. Some housekeeping proteins may be highly expressed in a particular cell type and they may be linear in sample amounts of up to 10 lg, while others may be linear up to 40 lg of the sample loaded for the same cell type [1]. Surprisingly, we found that actin was strongly upregulated upon treatment with EGF in A431 cells (Fig. 2), suggesting that actin is not suitable as a loading control in these cells. Similar results showing actin and other loading control proteins to be affected by culture conditions have been reported earlier [1,2]. This is a worrying example, indicating that housekeeping
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2. 5
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g ERK 1/2
Table 2 Variation of normalized tubulin signals for whole loading range between 4 and 20 lg for three operators and three membranes each. Operator
Tubulin Actin GAPDH
Normalized Cy3 tubulin/Cy5 total protein ratio (CV%)
1 2 3
Membrane 1
Membrane 2
Membrane 3
10.0 8.3 8.8
8.7 5.6 9.7
7.5 5.9 7.2
5 4 3
10
20
2
10 μg
20
A: Tubulin
Cy5
0 1.2
1
2
3
4
5
6
7
8
9 10
Cy3
1 0.8 0.6
0.2
12
0 2
1
2
3
4
5
6
7
8
9 10
1.5 1
C: GAPDH
0.5 0 1
2
3
4
5
6
7
8
9 10
Sample number Fig.4. Normalization of ERK1/2 signals using housekeeping protein normalization. Various amounts (2.5, 5, 10, 15, and 20 lg) of the same CHO cell lysate were loaded onto the gel in duplicate and subjected to Western blotting. ERK1/2 was targeted using anti-ERK1/2 primary antibody and Cy3-labeled secondary antibody. Tubulin, actin, and GAPDH control proteins were targeted using a mixture of their corresponding primary antibodies and Cy5-labeled secondary antibody in a multiplexed experiment on the same membrane. Normalized Cy3 ERK signals (Cy3/Cy5 ratios) were obtained after image analysis using (A) Cy5 tubulin, (B) Cy5 actin, or (C) GAPDH control signals and are presented in the bar graphs. CV% for the normalized ratio over the whole loading range was calculated for each loading control and presented in Table 1.
Table 1 Variation of normalized ERK1/2 signals for various sample loads. Loading control
Normalized ratio within 2.5–20 lg sample load (CV%)
Cy5 total protein Tubulin Actin GAPDH
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Loading errors were simulated by loading 2.5, 5, 10, 15, and 20 lg of the same CHO cell lysate. The ERK1/2 signals were normalized using Cy5 total protein or housekeeping proteins as control. CV% between the sample loads was calculated.
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proteins can be unexpectedly regulated in some experiments depending on the stimulus and cell type. Cy5 total protein prelabeling is unaffected by cell type and cellular treatments or culture conditions and relies on multiple protein signals and, therefore, it is a more reliable method. Total protein signal used for normalization can be obtained by prelabeling the sample. NHS prelabeling introduces a covalent bond between Cy5 and lysine residues or the N-terminus of proteins [9], enabling cotransfer along with unlabeled proteins to the membrane. Total protein signal from either gel or membrane will reflect the sample load, but using the signals on the membrane includes any transfer variation in the normalization. Prelabeling of
Cy3 ERK 1/2 signal (volume x 10-3)
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Sample number Fig.5. Comparison of target protein signals from unlabeled or Cy5-prelabeled cell lysate. Samples (10 and 20 lg) of unlabeled or Cy5-prelabeled CHO cell lysate were applied as indicated in triplicate on the same gel and subjected to Western blotting. The Cy3 signal intensity of ERK1/2 targeted by anti-ERK1/2 primary antibody and Cy3-labeled secondary antibody is presented in the bar graph.
cell lysates for total protein normalization is performed directly in the lysis buffer adjusted to the same final reaction volume for the samples to be compared. The labeling reaction is compatible with many common cell lysis buffers. The optimal pH is in the range of 8–9 [9], but lysis buffers with a pH range of 7–8 are also compatible and produce sufficient labeling for normalization purposes. The resolution and the labeling efficiency, negatively affected by suboptimal pH and diluted Cy5 dye in this protocol, do not affect normalization so long as the total signal is proportional and the labeling efficiency is the same for all samples that are being compared. We have shown that the labeling reaction is linear and proportional up to 80 and 300 lg of cell lysate proteins per reaction (20-ll reaction volume) for room temperature and +4 °C, respectively. The labeling reaction at room temperature is suitable for lower protein amounts (up to 80 lg per reaction) and incubation on ice is recommended for higher protein amounts or for temperature-sensitive samples (Fig. 1). The normalized results of an identical sample (i.e., the ratio between the target and the control signals) should remain constant regardless of the amount of loaded sample. A typical quantitative Western blot experiment involves the same sample type, treated or grown under different conditions, thus conferring variations on the sample load. In this study, we simulated loading errors by applying the same sample between 2.5 and 20 lg of total cell lysate protein. This broad range was chosen to compare the
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Fig.6. Analysis of PP2A levels in EGF-treated A431 cells using Cy5 total protein normalization. A431 cell lysates from control and EGF-treated cells were mixed with increasing amounts of treated lysate (0, 25, 50, 75, and 100% treated lysate) and prelabeled with Cy5. Samples (15 lg) were loaded in duplicates as indicated. After transfer, the membranes were probed with anti-PP2A primary antibody and Cy3-labeled secondary antibody. (A) Cy3 PP2A and (B) Cy5 total protein signals on the membrane and (C) normalized ratios were obtained after image analysis and are presented in bar graphs.
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Fig.7. Analysis of Smad2/3 levels in TGF-b-stimulated NMuMG cells using Cy5 total protein normalization. NMuMG cells were starved for 6 h followed by stimulation with TGF-b for 0, 30, or 60 min or 6 h. Untreated control cells, indicated by ‘‘C’’, were grown for 24 h. Cell lysates from the different samples were Cy5-prelabeled and 20 lg was loaded as indicated and subjected to Western blotting. Molecular weight markers are indicated by ‘‘M’’. The membrane was probed for Smad2/3 using anti-Smad2/3 primary antibody and Cy3-labeled secondary antibody. The Smad2 and Smad3 protein bands are indicated by arrows and the (A) Cy3 signals, (B) Cy5 total protein signals, and (C) normalized signals (Cy3/Cy5 ratios) were obtained after image analysis and are presented in the graphs.
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performance of normalization and robustness using different types of loading control signals. In this side-by-side evaluation of different normalization methods, Cy5 total protein normalization produced a stable target-to-control ratio over a wider range of sample concentrations (lower CV%), suggesting that it is a more
reliable alternative to using endogenous proteins as control (Figs. 3 and 4). GAPDH signals should not be used as loading control for the CHO cell type used in this study because they were not proportional to the protein load, producing no visible normalization
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effect. Actin and tubulin performed better above 10 lg of cell lysate protein. We show the robustness of the method by normalized tubulin signals; using Cy5 total protein as the control for a broad loading range is very reproducible between labeling reactions, operators, and membranes (Fig. 1 and Table 2), with CVs typically between 5 and 10%. An insufficient normalization may be due to large differences in the intercepts of target and control signals. It therefore is good practice to confirm linearity and proportionality of both target and control signals in a preexperiment using a dilution series of the cell lysate sample to be used. It may be necessary to optimize the probing conditions, such as antibody dilutions, to achieve linearity. Since Cy5 total protein detection is antibody independent, any variations from antibody quality or probing conditions can be avoided. It is also important to confirm that the background subtraction for both target and control is adequate. The prelabeling must not interfere with the binding of the primary antibody to its target. This was confirmed when the same signal intensity of the target was seen in both prelabeled and unlabeled samples (Fig. 5). The dye-to-protein ratio after labeling is dependent on the concentration of Cy5 dye in the reaction [9]. The degree of protein labeling using a protocol with diluted Cy5 dye reagent is low (below 5%). Most of the proteins remain unlabeled; hence the risk of interference from Cy5 molecules is minimal. We successfully applied total protein normalization to some typical Western experiments and could with high confidence analyze a 50% increase in PP2A and a 50% decrease in Smad2 target levels in A431 and NMuMG cells, respectively (Figs. 6 and 7). Using Cy5-prelabeled NMuMG cell lysate, we noticed a difference in total protein composition for the 0-min sample, which we would not have seen using a traditional Western blot with unlabeled samples (Fig. 7). A Cy5 total protein signal can also be used to confirm even loading and good gel separation results before transfer. Transfer quality can also be confirmed on the membrane using Cy5-prelabeled samples.
In conclusion, total protein normalization produces reliable quantitative results and there are many advantages to using Cy5 total protein as the loading control over a single housekeeping protein.
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Acknowledgments
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The NMuMG cell lysate samples were kindly provided by Professor Pontus Aspenström and Dr. Katarina Reis, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2015.06.017.
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References
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[1] S.L. Eaton et al., Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting, PLoS ONE 8 (2013) e72457. [2] A. Dittmer, J. Dittmer, b-Actin is not a reliable loading control in Western blot analysis, Electrophoresis 27 (2006) 2844–2845. [3] S. Greer et al., Housekeeping genes: expression levels may change with density of cultured cells, J. Immunol. Methods 355 (2010) 76–79. [4] M. Zellner et al., Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples, Electrophoresis 29 (2008). 3621–3362. [5] C. Welinder, L. Ekblad, Coomassie staining as loading control in Western blot analysis, J. Proteome Res. 10 (2011) 1416–1419. [6] A. Gurtler et al., Stain-free technology as a normalization tool in Western blot analysis, Anal. Biochem. 433 (2013) 105–111. [7] I. Romero-Calvo et al., Reversible Ponceau staining as a loading control alternative to actin in Western blots, Anal. Biochem. 401 (2010) 318–320. [8] K.A. Janes, An analysis of critical factors for quantitative immunoblotting, Sci. Signaling 8 (2015) rs2. [9] E.J. Bjerneld et al., Prelabeling of diverse protein samples with a fixed amount of Cy5 for sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. Anal. Biochem. 2015 (in press).
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Cy5 total protein normalization in Western blot analysis
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Åsa Hagner-McWhirter ⇑, Ylva Laurin, Anita Larsson, Erik Bjerneld, Ola Rönn
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GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden
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Article history: Received 12 March 2015 Received in revised form 10 June 2015 Accepted 11 June 2015 Available online xxxx Keywords: Western blotting Normalization Loading control CyDye Prelabeling Housekeeping protein
a b s t r a c t Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0 lg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence. Ó 2015 Elsevier Inc. All rights reserved.
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Western blotting is used for qualitative and quantitative analysis of proteins. Qualitative analysis may confirm the identity, presence, or absence of a protein or provide a rough estimate of the amounts of protein. Uneven sample loading is common owing to errors in protein quantitation, cell number estimation, or pipetting. Therefore, to quantitatively compare the levels of specific proteins between samples in Western blots, it is common to relate the target signal to a presumably constitutively expressed endogenous or a so-called housekeeping protein such as glyceraldehyde-3phosphate dehydrogenase (GAPDH)1 or structural proteins such as b-tubulin and b-actin. The signal of the housekeeping protein is expected to be proportional to total protein and hence the cell number loaded in each well. However, this method has many experimental pitfalls, such as cell- or tissue-dependent linear response range of the sample, suboptimal dilution, and/or poor quality of the antibodies, which may weaken the proportionality of signal-to-sample loading. In addition, the reliability of using housekeeping proteins as loading controls is under scrutiny because ⇑ Corresponding author. Fax: +46 18 6121844. E-mail address: [email protected] (Å. Hagner-McWhirter). 1 Abbreviations used: BSA, bovine serum albumin; PVDF, polyvinylidene difluoride; CHO, Chinese hamster ovary; PP2A, protein phosphatase type 2A; EGF, epidermal g r o w t h f a c t o r ; E R K , e x t r a c e l l u l a r s ig n a l - r e g u l a te d k i n a s e ; G A P D H , glyceraldehyde-3-phosphate dehydrogenase; NHS, N-hydroxysuccinimide; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; DTT, dithiothreitol; RuBPS, ruthenium(II)–tris(bathophenanthroline disulfonate).
culture conditions or treatments may affect the expression level of housekeeping proteins [1–3]. It is important to choose an appropriate method to detect the protein signal and it is critical to qualify target and control signals to be used for normalization. Housekeeping proteins are widely used but not always reliable as loading controls and an increased interest in using total protein instead has been seen in recent publications [1,4–7]. A fluorescence-based detection system produces stable signals, high sensitivity, broad dynamic range, and the ability to normalize by detecting several targets simultaneously by multiplexing on the same blot. Chemiluminescence detection requires more stringent experimental control, and the lack of multiplexing capability necessitates stripping and reprobing procedures that may increase the risk of experimental variation. Using the total protein signal from a sample lane for normalization has been investigated previously and shown to be the most reliable loading control. The total protein signal was obtained by poststaining the membrane with Ponceau S [7], RuBPS [4], and Coomassie [5] stains or detected with ultraviolet radiation [6]. We present a reliable and easy normalization method based on Cy5-prelabeled total protein. The labeling reaction is based on NHS chemistry with a ready-to-use Cy5 dye reagent [8]. The Cy5-labeled proteins are then transferred to the membrane followed by probing with primary antibody against target protein and Cy3-labeled secondary antibodies in a multiplex experiment on the same blot. The Cy5 total protein signal for each lane on
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the membrane is used as a control signal for normalization. We provide comparative performance evaluations of total protein normalization and endogenous protein normalization using housekeeping proteins, in addition to quantitative Western blot applications in which total protein was used for normalization.
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Materials and methods
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Total protein concentration was determined using a Coomassie Plus protein assay kit (Thermo Scientific). The Cy5 prelabeling reaction volume was adjusted to 19 ll with original lysis buffer for all the samples, and 1 ll of Amersham WB Cy5 dye reagent (approximately 250 pmol Cy5–NHS/ll; GE Healthcare), which had been freshly diluted 1:10 or 1:20 with ultrapure water just prior to labeling, was added to start the labeling reaction. The samples were mixed and incubated for 30 min at room temperature or on ice. The reactions were stopped by the addition of 20 ll of Amersham WB loading buffer (50 mM Tris–Cl, 0.25% (w/v) orange G, 4% (w/v) SDS, 0.5 mM lysine; GE Healthcare) containing 40 mM DTT. The reaction volumes were sometimes scaled up two- or fivefold to a final reaction volume of 40 or 100 ll. The samples were heated for 3–5 min at 95 °C and 20 ll of each sample was applied to the wells of an Amersham WB gel card 14, 8–18% (GE Healthcare). Amersham WB molecular weight markers (GE Healthcare) were diluted 1:60 or 1:80 in diluted loading buffer with 40 mM DTT (1 part loading buffer plus 1 part ultrapure water). We performed electrophoresis in an Amersham WB system (GE Healthcare), at 600 V for 45–50 min. The Cy5-prelabeled and unlabeled proteins were transferred to an Amersham PVDF card (GE Healthcare) using a tank transfer protocol and a transfer buffer (192 mM glycine, 25 mM Tris) containing 20% (v/v) ethanol at 100 V for 30 min. The Amersham WB system default probing protocol was used. The membranes were blocked in 3% (w/v) BSA in PBS-T or TBS-T (containing 0.1% (v/v) Tween 20) for 10 min, and a PBS-T or TBS-T buffer was used for the dilution and wash steps. PBS or TBS buffer was used for the final wash. The control and target primary antibodies were mixed in a total probing volume of 10 ml and incubated for 1–3 h followed by secondary antibody incubation for 30 min using Amersham WB Cy3- or Cy5-labeled secondary antibodies (GE Healthcare). The membranes were dried for 10 min in the system before scanning in the Cy3 and Cy5 channels of the Amersham WB system using automatic or manual settings. We used the Amersham WB evaluation software with built-in normalization to determine target, control signals, and normalized ratio. Coefficients of variation (CVs) were calculated using Microsoft Excel software. The background was subtracted from the target protein bands and Cy5-labeled total protein with rolling ball sizes of 400 and 1000 (default background subtractions), respectively.
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Linearity of Cy5 total protein labeling reaction
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Chinese hamster ovary (CHO) cells expressing monoclonal antibodies (suspension cell culture) were grown in a 50-L WAVE bag (working volume 25 L) on a WAVE bioreactor system (GE Healthcare). Cells were grown in ActiCHO medium (GE Healthcare) and from day 3 fed with ActiCHO Feed-A and ActiCHO Feed-B (GE Healthcare) daily. From day 6, the glucose concentration was measured and glucose administered to the culture up to 6 g/L daily. The cells were harvested by centrifugation at 3000g for 20 min at room temperature. The cell pellet was washed twice by resuspending the pellet in a fourfold volume of PBS buffer, followed by centrifugation (as above), and discarding the
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supernatant. The washed cell pellet was lysed for 1.5 h on ice with occasional mixing in 1 ml of Mammalian Protein Extraction Buffer (GE Healthcare) containing 10 ll/ml Protease Inhibitor Mix (GE Healthcare) per approximately 1 ml of cell pellet. Cell debris was removed by centrifugation at 16,000g for 30 min at +4 °C. The cell lysate supernatant was transferred to a new tube. The linearity of the labeling reaction was analyzed by incubating various amounts of total protein with the Cy5 dye reagent. Duplicate tubes containing 10, 20, 40, 80, 160, 240, and 300 lg of cell lysate per 20 ll were prepared and labeled with Cy5 dye reagent (which had been diluted 1:10) in equal final reaction volumes of 100 ll of lysis buffer. The samples described above were incubated at room temperature (approximately +22 °C) and at +4 °C (on ice) in parallel. The reactions were stopped by the addition of an equal volume (100 ll) of loading buffer. The samples were diluted 1:5 in diluted loading buffer (1 part loading buffer and 1 part ultrapure water) and 20-ll samples containing 1, 2, 4, 8, 16, 24, or 30 lg of CHO cell lysate were applied to the wells of the gel. The gels were scanned after electrophoresis, and the membranes were scanned after transfer in the Cy5 channel. Linearity for lower amounts of total proteins was investigated in a separate experiment (as above) with reactions containing 5, 10, 20, and 40 lg of CHO cell lysate. Samples (20 ll) without dilution were applied to the wells of the gels and scanned after electrophoresis in the Cy5 channel. The Cy5 total protein signal from each lane was plotted against the amount of protein.
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Detection of ERK1/2 in various amounts of cell lysate
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CHO cells were lysed as described above and a dilution series with incremental steps of 5 (from 5 to 40 lg per 20-ll reaction volume) was prepared in duplicate. The volume was adjusted to 95 ll with lysis buffer for all protein amounts, and 5 ll of Cy5 dye reagent (which had been diluted 1:20 in ultrapure water) was added to start the labeling reaction. The duplicate reactions were stopped by the addition of an equal volume (100 ll) of loading buffer and 20 ll of each sample, containing 2.5 to 20.0 lg of cell lysate, was loaded per well. The unlabeled samples were prepared in parallel but 5 ll of ultrapure water was added instead of the diluted Cy5 dye reagent and 20 ll of each sample was loaded per well. ERK1/2 was targeted by rabbit anti-mitogen-activated protein kinase (MAPK) primary antibody (Sigma–Aldrich) that had been diluted 1:5000 and Cy3-labeled (anti-rabbit IgG) secondary antibody, which had been diluted 1:2500. Endogenous control proteins were targeted in the unlabeled samples by mouse anti-tubulin and mouse anti-actin primary antibodies (both of which had been diluted 1:5000) and mouse anti-GAPDH primary antibody, which had been diluted 1:2500 (Sigma–Aldrich). After it was verified that there was no cross-reaction, the three mouse primary antibodies were mixed and incubated for 1.5 h at room temperature. This was followed by incubation with Cy5-labeled (anti-mouse IgG) secondary antibody, which had been diluted 1:2500, for 1 h at room temperature. The membranes were dried and scanned in the Cy3 and Cy5 channels. The ERK1/2 signals were normalized against Cy5 total protein or Cy5 tubulin, actin, or GAPDH endogenous protein signals.
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Reproducibility of Cy5 total protein normalization
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CHO cell lysates were prepared as described above and three different operators performed Cy5 prelabeling of 8, 24, and 40 lg in quadruplicate reactions using Cy5 dye reagent diluted 1:10. The reactions were stopped with an equal volume of loading buffer and heated as described above. The samples were subjected to
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Western blotting using three different membranes per operator. Tubulin was targeted with mouse anti-tubulin (Sigma–Aldrich), which had been diluted 1:1000, and Cy3-labeled (anti-mouse IgG) secondary antibody, which had been diluted 1:2500. The dried membranes were scanned in the Cy3 and Cy5 channels. The Cy3 tubulin signals were normalized using Cy5 total signal and the CV of the normalized ratio (Cy3/Cy5) between 4 and 20 lg was calculated for each operator and membrane.
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Detection of ERK1/2 in Cy5-prelabeled and unlabeled sample
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CHO cells were lysed as described above and samples containing 20 or 40 lg per 20-ll volume were prepared in duplicate. Cy5 dye reagent (diluted 1:10) was used to prelabel one sample set in a final volume reaction volume of 100 ll lysis buffer. An equal volume (100 ll) of loading buffer containing 40 mM DTT was added to both the Cy5-prelabeled (to stop the reaction) and the unlabeled samples; 20-ll samples containing 10 or 20 lg of CHO cell lysate were applied in triplicate to the same SDS–PAGE gel. Rabbit anti-MAPK primary antibody (Sigma–Aldrich) was diluted 1:5000 and Cy3-labeled (anti-rabbit IgG) secondary antibody was diluted 1:2500 and used to target ERK1/2. The dried membranes were scanned in the Cy3 and Cy5 channels. The target Cy3 signal intensity of ERK1/2 was compared for Cy5-prelabeled and unlabeled samples.
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Detection of actin and phospho-protein phosphatase type 2A (PP2A) in epidermal growth factor (EGF)-stimulated A431 cells
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Cell lysates (2.5 lg/ll) of A431 (human epidermal carcinoma cell line) cells stimulated with EGF were purchased from Santa Cruz Biotechnology. Lysates of unstimulated A431 (control) cells were mixed with increasing amounts (0, 25, 50, 75, and 100%) of EGF-stimulated cell lysates and prelabeled with diluted (1:10) Cy5 dye reagent. Duplicate samples containing 15 lg (actin detection) or 20 lg (PP2A detection) of the cell lysate were loaded onto the SDS–PAGE gel. One blot was probed for the detection of actin with mouse anti-actin primary antibody (Sigma–Aldrich) diluted 1:5000 and Cy3-labeled antibody (anti-mouse IgG) diluted 1:2500. Another blot was probed for the detection of PP2A using rabbit anti-PP2A primary antibody (C subunit (52F8); Cell Signaling), which had been diluted 1:750, and Cy3-labeled (anti-rabbit IgG) secondary antibody, which had been diluted 1:2500. The dried membranes were scanned in the Cy3 and Cy5 channels. The actin and PP2A signals were normalized against Cy5 total protein on the two separate blots.
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Detection of Smad2/3 in NMuMG cells
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NMuMG cells (mouse mammary epithelial cell line) were grown in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 0.1% insulin. The cells were starved overnight (16 h), followed by treatment with 1 ng/ml tumor growth factor-b (TGF-b) for 0, 15, 30, or 60 min or 6 h. Untreated cells (control) were grown without starvation for 24 h. The cells were washed with PBS and detached with a rubber policeman in lysis buffer containing 1% Triton X-100. The NMuMG cell lysates were prelabeled with diluted (1:10) Cy5 dye reagent. Samples containing 20 lg of the cell lysate were subjected to Western blot analysis and probed as described above with the exception that the primary antibody was incubated for 3 h instead of 1 h. TBS-T or TBS buffer was used for the dilution and wash steps. Smad2/3 was detected using mouse anti-Smad2/3 (C-8; Santa Cruz Biotechnology) diluted 1:250 and Cy3-labeled (anti-mouse IgG) secondary antibody, which had been diluted
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1:2500. The membrane was dried and scanned in the Cy3 and Cy5 channels. The Smad2/3 signals were normalized against Cy5 total protein.
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Results
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Linearity of Cy5 prelabeling of CHO cell lysates
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The signal response of labeled total protein was tested for a broad range of sample concentrations at both room temperature and +4 °C (Fig. 1A and B). The Cy5 prelabeling reaction was linear up to 80 lg per reaction (reaction volume 20 ll, corresponding to 4 lg total protein/ll) for reactions performed at room temperature and linear across the whole tested range up to 300 lg per reaction (corresponding to 15 lg total protein/ll) when the reaction was performed at +4 °C (Fig. 1C). The linearity at lower amounts of total protein was confirmed at room temperature and +4 °C (Fig. 1D). The variation in signal intensity between replicate reactions was low (Fig. 1D). The results in Fig. 1 are based on analysis in the gel, and similar results were obtained by analysis of the signals after transfer to the membrane (data not shown).
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Actin versus total protein as loading control in EGF-stimulated cells
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The same amounts of Cy5-prelabeled A431 cell lysate mixtures with increasing concentrations of EGF-stimulated lysate were subjected to Western blotting targeting the housekeeping protein actin with anti-actin primary antibodies and Cy3-labeled secondary antibodies. The Cy3 actin signals increased (Fig. 2A), but the Cy5 total protein signal was unaffected, indicating similar loading of the samples (Fig. 2B). Normalization to Cy5 total protein reveals that actin levels increased dramatically after EGF treatment (Fig. 2C). Actin was regulated by EGF treatment and thus inadequate as a loading control for this cell type and treatment, whereas the more reliable Cy5 total signal would accurately reflect the sample load.
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Quantitation of ERK1/2—comparing normalization performance using endogenous control proteins or total protein
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To compare quantitation of the target over a wide loading range, a dilution series of the same CHO cell lysate was used. To measure the degree of normalization efficiency, we calculated the CV% of the normalized ratio across the loading range. The ERK1/2 target signals from CHO cell lysates produced a proportional response to sample load, and variation between the duplicate samples was very low for all blots (Figs. 3 and 4). The ERK1/2 signals were normalized using either Cy5 total signal within the whole lane (Fig. 3) or one of the endogenously expressed loading control proteins actin, tubulin, or GAPDH (Fig. 4). The Cy5 total protein control signals showed low variation between duplicate labeling reactions and the total signal was proportional to sample load (Fig. 3B). A sample load of 20 lg gave a lower Cy5 total signal, but the same was observed for the Cy3– ERK1/2 antibody-mediated signal, suggesting that less sample was loaded in these wells and the reduction was not related to the Cy5 prelabeling. Normalization of ERK1/2 signals using Cy5 total protein resulted in similar target/control ratios with a CV of 7% over the whole sample loading range (Fig. 3C and Table 1). Normalization of ERK1/2 signals using tubulin, actin, or GAPDH instead resulted in ratios with a CV of 18, 13, and 54%, respectively (Fig. 4 and Table 1). Using tubulin or actin as control resulted in a higher variation of normalized values across the whole range with a visible difference in ratio between high and low sample amounts (Fig. 4A and B). GAPDH showed very poor performance since the
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Fig.1. Linearity of Cy5 total protein prelabeling. Duplicate Cy5 prelabeling reactions containing 10, 20, 40, 80, 160, 240, and 300 lg CHO cell lysate per reaction as indicated were incubated at (A) room temperature (RT) or at (B) +4 °C for 30 min, and fivefold-diluted samples were subjected to electrophoresis and scanning using the Amersham WB system. (C) The Cy5 total signal within the whole lane from the gels in (A) and (B) was plotted against the amount of protein in the reaction for room temperature (filled circles) and +4 °C (open circles) incubation. (D) Triplicate Cy5 prelabeling reactions containing 5, 10, 20, or 40 lg CHO cell lysate per reaction were incubated at room temperature (filled circles) or at +4 °C (open circles) for 30 min and the Cy5 total signal within the whole lane was plotted against the amount of protein in the reaction.
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Fig.2. Activation of actin in response to EGF treatment. A431 cell lysates from control and EGF-treated cells were mixed with increasing amounts of treated lysate (0, 25, 50, 75, and 100% treated lysate), prelabeled with Cy5, and loaded in duplicates as indicated. After transfer, the membranes were probed for b-actin using anti-actin primary antibody and Cy3-labeled secondary antibody. (A) Cy3 b-actin signals, (B) Cy5 total protein, and (C) normalized Cy3/Cy5 ratio obtained after image analysis are presented in the bar graphs. 326 327 328 329
specific signal did not increase much with increasing sample load and showed almost no normalization effect, resulting in a large variation in normalized ratio over the sample range (Fig. 4C and Table 1).
Reproducibility of Cy5 total protein normalization
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Using Cy5 total protein normalization, three different operators performed Western blotting targeting tubulin using primary
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Fig.3. Normalization of ERK1/2 signals using total protein normalization. Various amounts of the same CHO cell lysate were Cy5-prelabeled in duplicate reactions. Samples containing 2.5, 5, 10, 15, and 20 lg total protein were applied to the gel as indicated. The prelabeled samples were subjected to Western blotting and the membrane was probed with anti-ERK1/2 primary antibody and Cy3-labeled secondary antibody. (A) Cy3 ERK signal, (B) Cy5 total signal within each lane, and (C) the normalized ratio of Cy3/ Cy5 obtained after image analysis are presented in the bar graphs.
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antibody and Cy3-labeled secondary antibody. For the whole sample loading range (4–20 lg) in quadruplicate reactions and all three operators and triplicate membranes, the normalized ratio had an average CV of 8% (Table 2), showing that the normalization method was very reproducible.
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Effect of Cy5 prelabeling on target signal intensity
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To investigate whether Cy5 prelabeling adversely affected antibody binding to its target, we compared ERK1/2 signals from unlabeled and Cy5-prelabeled CHO cell lysate that had been applied to the same SDS–PAGE gel. Half of the gel was loaded with Cy5-prelabeled cell lysate and the other half with unlabeled samples. The Western blot membrane was incubated with the same antibody solutions and imaged with the same settings. Triplicate samples of 10 and 20 lg of CHO cell lysate were compared under identical conditions. No significant differences in signal intensities were seen, showing that Cy5 prelabeling had no effect on the detection of ERK1/2 (Fig. 5). Using this labeling protocol, the proportion of Cy5-labeled proteins is below 5% (data not shown), leaving the majority unlabeled. This suggests that antibody binding is not likely to be affected by Cy5 prelabeling of samples.
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Analysis of PPA2 and Smad2/3 levels using Cy5 total protein normalization To demonstrate the accuracy of analysis using total protein normalization we studied the effect of EGF treatment on the expression levels of PP2A in A431 cell lysates. Cy5-prelabeled cell lysate samples with various proportions of EGF-stimulated cell lysate were subjected to Western blotting. The PP2A signals were increased with EGF treatment (Fig. 6A), and the Cy5 total protein signals were similar, indicating low variation in sample load (Fig. 6B). When the PP2A signals were normalized using Cy5 total protein as a control, the results showed that EGF stimulation caused an approximately 50% upregulation of PP2A (Fig. 6B). In
another experiment, regulation of Smad2 and Smad3 by TGF-b treatment for various lengths of time was analyzed by Western blotting using Cy5 total protein normalization. The signal intensities of Smad2 without normalization showed a downregulation (Fig. 7A). Fig. 7B shows the sample load of the various samples, and when Smad2 was normalized to Cy5 total protein a clear effect was confirmed as a result of TGF-b treatment, showing an approximately 50% downregulation at 30 and 60 min (Fig. 7C). A small difference in Smad2 level between 30 and 60 min could also be seen. Smad3 was not changed as a result of TGF-b treatment incubation time (data not shown). The control sample (0 h incubation) had a different protein composition compared to the other samples, with a high expression of proteins with molecular weights ranging between 55 and 65 kDa (Fig. 7B).
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Discussion
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The Western blotting work flow contains many steps that potentially can affect the data quality, and one of the most important factors is the normalization [8]. Normalization of target signals using housekeeping proteins or total protein signals is critical to minimize the adverse effects of loading variability in quantitative Western blotting. The sources of loading variability include errors in protein quantitation of different samples or the estimation of sample amounts (e.g., one culture dish per sample). Single endogenous loading controls are often used routinely without confirming their normalization performance. Some housekeeping proteins may be highly expressed in a particular cell type and they may be linear in sample amounts of up to 10 lg, while others may be linear up to 40 lg of the sample loaded for the same cell type [1]. Surprisingly, we found that actin was strongly upregulated upon treatment with EGF in A431 cells (Fig. 2), suggesting that actin is not suitable as a loading control in these cells. Similar results showing actin and other loading control proteins to be affected by culture conditions have been reported earlier [1,2]. This is a worrying example, indicating that housekeeping
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Table 2 Variation of normalized tubulin signals for whole loading range between 4 and 20 lg for three operators and three membranes each. Operator
Tubulin Actin GAPDH
Normalized Cy3 tubulin/Cy5 total protein ratio (CV%)
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Sample number Fig.4. Normalization of ERK1/2 signals using housekeeping protein normalization. Various amounts (2.5, 5, 10, 15, and 20 lg) of the same CHO cell lysate were loaded onto the gel in duplicate and subjected to Western blotting. ERK1/2 was targeted using anti-ERK1/2 primary antibody and Cy3-labeled secondary antibody. Tubulin, actin, and GAPDH control proteins were targeted using a mixture of their corresponding primary antibodies and Cy5-labeled secondary antibody in a multiplexed experiment on the same membrane. Normalized Cy3 ERK signals (Cy3/Cy5 ratios) were obtained after image analysis using (A) Cy5 tubulin, (B) Cy5 actin, or (C) GAPDH control signals and are presented in the bar graphs. CV% for the normalized ratio over the whole loading range was calculated for each loading control and presented in Table 1.
Table 1 Variation of normalized ERK1/2 signals for various sample loads. Loading control
Normalized ratio within 2.5–20 lg sample load (CV%)
Cy5 total protein Tubulin Actin GAPDH
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Loading errors were simulated by loading 2.5, 5, 10, 15, and 20 lg of the same CHO cell lysate. The ERK1/2 signals were normalized using Cy5 total protein or housekeeping proteins as control. CV% between the sample loads was calculated.
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proteins can be unexpectedly regulated in some experiments depending on the stimulus and cell type. Cy5 total protein prelabeling is unaffected by cell type and cellular treatments or culture conditions and relies on multiple protein signals and, therefore, it is a more reliable method. Total protein signal used for normalization can be obtained by prelabeling the sample. NHS prelabeling introduces a covalent bond between Cy5 and lysine residues or the N-terminus of proteins [9], enabling cotransfer along with unlabeled proteins to the membrane. Total protein signal from either gel or membrane will reflect the sample load, but using the signals on the membrane includes any transfer variation in the normalization. Prelabeling of
Cy3 ERK 1/2 signal (volume x 10-3)
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Sample number Fig.5. Comparison of target protein signals from unlabeled or Cy5-prelabeled cell lysate. Samples (10 and 20 lg) of unlabeled or Cy5-prelabeled CHO cell lysate were applied as indicated in triplicate on the same gel and subjected to Western blotting. The Cy3 signal intensity of ERK1/2 targeted by anti-ERK1/2 primary antibody and Cy3-labeled secondary antibody is presented in the bar graph.
cell lysates for total protein normalization is performed directly in the lysis buffer adjusted to the same final reaction volume for the samples to be compared. The labeling reaction is compatible with many common cell lysis buffers. The optimal pH is in the range of 8–9 [9], but lysis buffers with a pH range of 7–8 are also compatible and produce sufficient labeling for normalization purposes. The resolution and the labeling efficiency, negatively affected by suboptimal pH and diluted Cy5 dye in this protocol, do not affect normalization so long as the total signal is proportional and the labeling efficiency is the same for all samples that are being compared. We have shown that the labeling reaction is linear and proportional up to 80 and 300 lg of cell lysate proteins per reaction (20-ll reaction volume) for room temperature and +4 °C, respectively. The labeling reaction at room temperature is suitable for lower protein amounts (up to 80 lg per reaction) and incubation on ice is recommended for higher protein amounts or for temperature-sensitive samples (Fig. 1). The normalized results of an identical sample (i.e., the ratio between the target and the control signals) should remain constant regardless of the amount of loaded sample. A typical quantitative Western blot experiment involves the same sample type, treated or grown under different conditions, thus conferring variations on the sample load. In this study, we simulated loading errors by applying the same sample between 2.5 and 20 lg of total cell lysate protein. This broad range was chosen to compare the
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Fig.6. Analysis of PP2A levels in EGF-treated A431 cells using Cy5 total protein normalization. A431 cell lysates from control and EGF-treated cells were mixed with increasing amounts of treated lysate (0, 25, 50, 75, and 100% treated lysate) and prelabeled with Cy5. Samples (15 lg) were loaded in duplicates as indicated. After transfer, the membranes were probed with anti-PP2A primary antibody and Cy3-labeled secondary antibody. (A) Cy3 PP2A and (B) Cy5 total protein signals on the membrane and (C) normalized ratios were obtained after image analysis and are presented in bar graphs.
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Fig.7. Analysis of Smad2/3 levels in TGF-b-stimulated NMuMG cells using Cy5 total protein normalization. NMuMG cells were starved for 6 h followed by stimulation with TGF-b for 0, 30, or 60 min or 6 h. Untreated control cells, indicated by ‘‘C’’, were grown for 24 h. Cell lysates from the different samples were Cy5-prelabeled and 20 lg was loaded as indicated and subjected to Western blotting. Molecular weight markers are indicated by ‘‘M’’. The membrane was probed for Smad2/3 using anti-Smad2/3 primary antibody and Cy3-labeled secondary antibody. The Smad2 and Smad3 protein bands are indicated by arrows and the (A) Cy3 signals, (B) Cy5 total protein signals, and (C) normalized signals (Cy3/Cy5 ratios) were obtained after image analysis and are presented in the graphs.
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performance of normalization and robustness using different types of loading control signals. In this side-by-side evaluation of different normalization methods, Cy5 total protein normalization produced a stable target-to-control ratio over a wider range of sample concentrations (lower CV%), suggesting that it is a more
reliable alternative to using endogenous proteins as control (Figs. 3 and 4). GAPDH signals should not be used as loading control for the CHO cell type used in this study because they were not proportional to the protein load, producing no visible normalization
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effect. Actin and tubulin performed better above 10 lg of cell lysate protein. We show the robustness of the method by normalized tubulin signals; using Cy5 total protein as the control for a broad loading range is very reproducible between labeling reactions, operators, and membranes (Fig. 1 and Table 2), with CVs typically between 5 and 10%. An insufficient normalization may be due to large differences in the intercepts of target and control signals. It therefore is good practice to confirm linearity and proportionality of both target and control signals in a preexperiment using a dilution series of the cell lysate sample to be used. It may be necessary to optimize the probing conditions, such as antibody dilutions, to achieve linearity. Since Cy5 total protein detection is antibody independent, any variations from antibody quality or probing conditions can be avoided. It is also important to confirm that the background subtraction for both target and control is adequate. The prelabeling must not interfere with the binding of the primary antibody to its target. This was confirmed when the same signal intensity of the target was seen in both prelabeled and unlabeled samples (Fig. 5). The dye-to-protein ratio after labeling is dependent on the concentration of Cy5 dye in the reaction [9]. The degree of protein labeling using a protocol with diluted Cy5 dye reagent is low (below 5%). Most of the proteins remain unlabeled; hence the risk of interference from Cy5 molecules is minimal. We successfully applied total protein normalization to some typical Western experiments and could with high confidence analyze a 50% increase in PP2A and a 50% decrease in Smad2 target levels in A431 and NMuMG cells, respectively (Figs. 6 and 7). Using Cy5-prelabeled NMuMG cell lysate, we noticed a difference in total protein composition for the 0-min sample, which we would not have seen using a traditional Western blot with unlabeled samples (Fig. 7). A Cy5 total protein signal can also be used to confirm even loading and good gel separation results before transfer. Transfer quality can also be confirmed on the membrane using Cy5-prelabeled samples.
In conclusion, total protein normalization produces reliable quantitative results and there are many advantages to using Cy5 total protein as the loading control over a single housekeeping protein.
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Acknowledgments
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The NMuMG cell lysate samples were kindly provided by Professor Pontus Aspenström and Dr. Katarina Reis, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2015.06.017.
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References
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