E/ec/rophoresrs 1990. I / , 581-588
Peter Jungblut Christoph Eckerskorn2 Friedrich Lottspeich2 Joachim Klose 'Institut fiir Humangenetik,Institut fur Toxikologie und Embryonalpharmakologie,Freie Universitat Berlin 2Max-Planck-Institutfur Biochemie, Genzentrum, Martinsried
Blotting efficiency of proteins on hydrophobic membranes
58 1
Blotting efficiency investigated by using two-dimensional electrophoresis, hydrophobic membranes and proteins from different sources Purification and chemical characterization of proteins may be achieved by combining two-dimensional electrophoresis (2-DE) and microsequencing or amino acid analysis. To enable this combination, the protein has to be transferred as completely as possible from the gel into the sequencer. In this study hydrophobic membranes were used as support for the transfer and proteins were transferred from the gels onto the membranes by semidry blotting. Blotting conditions were optimized to obtain high blotting efficiencies for as many proteins of a complex 2-DE pattern as possible. Under optimized conditions, blotting efficiencies between 60 % and 100 % were obtained for five marker proteins; the mean values from four regions of a 2-DE pattern from 29 unknown proteins of a complex protein mixture from mouse brain were between 60 % and 79 %. The four commercially available hydrophobic membranes that were compared showed only slight differences in protein amount on the membranes after blotting for whole protein patterns, whereas single proteins occurred with higher amounts on either one or the other membrane. The results ofthe blotting optimization allowed us to suggest a blotting mechanism with which systematic improvement of the blotting conditions is possible for problematic proteins.
1 Introduction Two-dimensional gel electrophoresis (2-DE) using the combination of isoelectric focusing (IEF) and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis [ I , 21 is the method with the highest resolution in protein analysis. At present, N-terminal sequencing methods are sensitive enough to sequence pmole amounts of proteins. With the development of membranes which can immobilize proteins blotted from polyacrylamide gels and that are directly applicable to the sequencing machine, 2-DE and microsequencing could be combined. The use ofone and the same support for blotting as well as for sequencing prevents loss and chemical modifications of the protein. Vandekerckhove et al. 131 and Aebersold et al. [41 developed positively charged membranes, while Matsudaira (51 and Eckerskorn et al. 161 introduced hydrophobic membranes for the immobilization of the proteins. The two main advantages of hydrophobic membranes are higher protein binding capacities and easy detection of proteins with commercially available protein dyes. Positively charged as well as hydrophobic membranes were used for blotting and sequencing ofproteinsfrom 2-DEgels [3-5,7,81, and anumber of proteins in the 2-DE patterns were identified by sequence comparison with the help of a sequence data base (71. To be able to also sequence proteins which occur in low amounts on the membrane after Coomassie Brilliant Blue staining, or which require the analysis ofinternal sequences [9,101, ahigh blotting efficiency is a prerequisite. In this investigation an attempt was made to find optimum conditions for blotting complex 2-DE protein patterns using
Correspondence: Dr. Peter Jungblut, Institut fur Toxikologie und Embryonalpharmakologie, Freie Universitat Berlin, Garystr. 5 , D-1000 Berlin 3 3 , Federal Republic of Germany Abbreviations: 2-DE, two-dimensional gel electrophoresis; f3-LG,bovine P-lactoglobulin; Be, blotting efficiency; BSA, bovine serum albumin; CA bovine carbonic anhydrase; IEF, isoelectric focusing;P,, protein remaining in the gel during blotting, RNase, bovine ribonuclease; SDS, sodium dodecyl sulfate; TI, soybean trypsin inhibitor
0VCH Verlagsgesellschaft mbH D-6940 Weinheim, 1990
the semidry blotting procedure. Compared to the conventional vertical blots in buffer tanks, semidry blotting has the advantages of reduced buffer consumption, minimized reactive impurities, reduced risk of formation of air bubbles due to a straightforward assembly, and reduced transfer time and heat development. On the basis of the results, a blotting mechanism for semidry blotting was suggested which could improve the blotting efficiency. Automatic evaluation ofthe 2D E patterns was used to compare different commercially available hydrophobic membranes.
2 Materials and methods 2.1 Protein sample preparation Solubilized proteins were extracted from the brains and embryos of mice, strain DBA/2J. Four brains were washed with 0.9 % NaCl to remove blood. After freezing and thawing, the brains were homogenized (five up-and-down strokes) in a 5 mL homogenizer in one volume of distilled water containing protease inhibitors. The final concentrations of the inhibitors were 5 mM ethylenediaminetetraacetate (EDTA), I mM phenylmethylsulfonyl fluoride (PMSF) and 0.1 pg pepstatin A (all from Sigma, Taufkirchen, FRG). The homogenate was placed into six tubes and centrifuged in a Beckman TI-50 rotor for 40 min at 225 000 g. The supernatants were combined, vortexed, and frozen in 200 pL aliquots. Each of the ten aliquots obtained contained 7.1 mg/mL protein as determined by the method of Peterson [ 1I]. A portion ofthe brain proteins was iodinated by the method described by Salacinski et al. [ 121. Twenty pL of the protein preparation were incubated for 15 min together with a chloramine T-bead (Iodobeads, Pierce, Oud-Beijerland, The Netherlands) and with NalZ5J,the radioactivity of which was 0.4 mCi. The iodinated proteins were separated from the free 25J-bygel filtration on aSephadex G25 (Pharmacia-LKB, Uppsala, Sweden) column. Marker proteins were also used in this study and iodinated as well. The marker protein mixture contained 2 pg each of bovine serum albumin (BSA, Sigma), bovine P-lactoglobulin (P-LG, Sigma), bovine ribonuclease (RNase, Sigma), bovine carbonic 0173-0835/90/0707-058 I $3.50+.25/0
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anhydrase (CA, Serva, Heidelberg, F R G ) and soybean trypsin inhibitor (TI, Sigma), solubilized together in 20 pL of a buffer containing 4 mM KH,PO,, 10 mM Na,HPO, and 120 mM NaC1. The marker proteins RNase, P-LG, TI, C A and BSA have the following molecular weights and p l values: 13 600, 7.8; 18 400,5.3; 20 100,4.5; 29 000,5.91;and 66 300,4.8, respectively, The mixture (20 pL) was iodinated with 0.25 mCi NaI2'J as described above. The radioactiivity of the '25J-labeled marker proteins obtained was 77 000 cpm/pL, that of the labeled brain proteins 200 000 cpm/pL. Solubilized proteins from mouse embryos were prepared and labeled with I4Camino acids as described by Klose [ 131. IJrea (Bio-Rad, Richmond, CA, USA), mercaptoethanol (Merck, Darmstadt, FRG) and Servalyt carrier ampholytes p H 5-7 (Serva) were added to all the protein preparations to obtain final concentrations of 9 M, 5 % v/v and 2 % v/v, respectively. The resulting solutions were used as protein samples for 2-DE.
2.2 2-DE The 2-DE technique first described by Klose [ 21 and modified as described by Jungblut and Seifert [ 141 was used to separate the proteins. The radioactivity ofthe sample applied to the IEF gels was determined and amounted to 801 000 cpm for marker proteins, 400 000 cprn for brain proteins and 125 000 cpm for the embryo proteins. Parameters influencing the blotting efficiency are the acrylamide concentration (the porosity) of the SDS gel and the ionic strength in that gel after 2-DE. The acrylamide concentration was 15 % w/v acrylamide, 0.2 % w/v N,N'-methylenebisacrylamide(1 5.2 % T, 1.3 % C). The ionic strength in the gel after 2-DE shoulld correspond to that ofthe electrode buffer,which was 25 mM Tris, 192 mMglycine and 0.1 % w/v SDS. The size of the slab gels was 6.5 (running direction) x 8.5 x 0.15 cm. To prevent loss of proteins by diffusion, the SDS gels were not equilibrateld with blotting buffer but were directly subjected to the blotting procedure.
2.3 Electroblotting Electroblotting was performed by the semidry procedure using a Sartoblot I1 blotting apparatus (Slartorius, Gottingen, FRG) with graphite electrodes. In a preceding investigation mouse brain proteins were blotted onto the siliconized glass fiber membrane Glassybond (Biometra, Gottingen, F R G ) or onto the polyvinylidene difluoride membrane Immobilon (Millipore, Bedford, MA, USA). The proteins were detected with Coomassie Brilliant Blue R-250 as described 171. The blotting parameters, i. e. the composition (ionic strength, pH, methanol and SDS content) of the blotting buffer. blotting time and current/cm2, were varied. The protein patterns obtained on the membranes were examinedl visually with regard to completeness in spot composition and with regard to spot intensities. With this more qualitative approach higher intensities of protein spots on the membrane were obtained with a blotting buffer containing 50 mM borate, p H 9.0, and 20 % methanol by blotting with 1 mA/cm2 for 3 h. The question of whether these conditions were optimal for the blotting efficiency was tested by quantitative deteirmination ofthe blotting efficiency, which was performed with the Glassybond membrane. The membrane was washed for about 30 s in acetone and then equilibrated in blotting buffer for 20 min with three changes. The blotting buffer contained borate and methanol in different concentrations witlh varying pH values.
Electrophoresis 1990, 11, 581-588
The effect of different SDS concentrations in the cathodic blotting buffer was also investigated. Filter papers (Nr. 2668/ ~ 2 4 9 3 4 Schleicher , and Schuell, Dassel, FRG) were washed for 30 rnin with three changes. The graphite electrodes were soaked with distilled water before setting up the blotting sandwich. The lower electrode was used as anode and the sandwich was built up from the anodic side as follows: the membrane waslaidonto threelayersoffi1terpaper:thegelwas transferred onto the membrane and overlaid with three layers of filter paper. Air bubbles between the layers were removed by gently rolling a glass rod over each layer but not over the membrane. Electrotransfer was performed at different levels of constant current. Blotting time was varied between 0.5 and 5 h. For the comparison of hydrophobic membranes, four membranes were used: Glassybond, Immobilon, another polyvinylidene difluoride membrane ProBlott (Applied Biosystems, Foster City, CA, USA) and a polypropylene membrane Selex-20 (Schleicher and Schuell, Dassel, FRG). Glassybond was prewetted in acetone and washed with blotting buffer as described above. Immobilon was washed for 10 rnin in methanol and then equilibrated in blotting buffer for 20 min with three changes. Selex-20 and ProBlott membranes were washed with methanol for 10 rnin and then used directly to set up the blotting sandwich as described for the Glassybond and Immobilon membrane.
2.4 Detection of proteins on membranes and gels Radioactively labeled proteins were detected by autoradiography. The gels were dried before autoradiography as described by Klose 131. The membranes were air-dried at room temperature for a few hours, Dried gels or membranes were laid on Kodak X-Omat A R 5 X-ray film and exposed for 2-5 days.
2.5 Automatic evaluation For quantitative investigations of protein patterns an automatic evaluation that included a video camera and a computer was performed as described by Prehm et al. [ 151. Optical densities were measured and care was taken that none ofthe spots selected had an intensity reaching the saturation level of the blackness of the film.
2.6 Determination of blotting efficiency To confirm the results of our qualitative investigation of the blotting conditions we developed a quantitative test which defines blotting efficiency by the equation
B , is the blotting efficiency, mmembrane is the amount of protein on the membrane after blotting and ingelis the amount of protein in the gel before blotting. The amount of protein in the gel cannot be determined directly, but both mgeiand mmembranemaY be determined indirectly by measuring the optical densities obtained by the use of radioactively labeled proteins and autoradiography. In this way the blotting efficiency can be determined directly from the measured optical densities:
Blotting efficiency of proteins on hydrophobic membranes
Electrophoresis 1990,II, 581-588
ODmembrane is the optical density of the protein investigated in the membrane and ODgelisthe optical density oftheproteininvestigated in the gel before blotting. When using Eq. (2) one has to consider the possibility that the radioactivity may be quenched differently by dried gels and membranes. No quenching effects occur with lZ5J-labeledproteins, and, therefore, '25J-labeled marker proteins were used to quantify the blotting efficiency under different blotting conditions. For the determination of ODgelit is not possible to use the same gel from which the proteins are blotted onto the membrane. Therefore, pattern comparison has to be performed on parallel gels. To minimize differences between the two parallel gels due to problems in reproducibility of 2-DE, the ODgeIvalues were calculated as a mean value from six gels. Another way to determine Beis to measure the O D of the proteins possibly left on the gel after blotting (ODblotged,and to set the sum of ODblotgeland ODmembraneequal 100 %. Beisthencalculated by
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thus leading to more precise results. Therefore, if protein loss during the blotting procedure can be excluded, Eq. (3) should be used. If a blotting efficiency below 100 %was obtained, the question was whether the protein was not transferred from the gel, or whether the protein has passed the membrane. In this case, ODblotgel was determined and the percentage of the protein left in the gel P, was calculated by Eq. (4).
P,=
ODbIotgel X 100
(4)
ODgeI For OD,,I a mean value of six analyses was used.
3 Results 3.1 Blotting eaciency of marker proteins
(3) In this case it is assumed that no protein was lost during the blotting procedure. The calculation of Bewith Eq. (3) has the advantage that no OD-values of parallel gels have to be used,
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In Fig. 1 the effect of varying blotting time, current/area, ionic strength, methanol concentration, pH, and SDS concentration on the blotting efficiency for Glassybond membranes is shown. The blotting efficiency of marker proteins was determined by quantitative analysis. The optimum conditions ob-
10
0
0.5
0.1
505-concmtration I%l Icathdd huffcrl
5u)hde
ZOrncdC
Figure 1. Blotting efficiency of '25J-labeled marker proteins. RNase (closed squares), trypsin inhibitor (open squares), P-LG (triangles), CA (open circles), and BSA (closed circles) were separated by 2-DE and blotted onto Glassybond membranes. The blotting buffer consisted of 50 mM borate, pH 9.0,20 %methanol. Proteins were blotted with 1 mA/cm2 for 3 h. One of these parameters was varied in each of the six diagrams (a)-(0. Proteins were detected by autoradiography. Blotting efficiency was determined in (a) and for BSA (all diagrams except c, 5 mM with Eq. (3). For the other proteinsindiagram (b)-(0Eq. (2)was used.
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Eleclrophoresis 1990, 11, 5XI-5XX
P. Jungblut el a!.
tained by the preceding qualitative investigation as described in Section 2.3 were used. Only one parameter was varied in each investigation. The blotting efficiency depends on the blotting time (Fig. la) and was calculated according to Eq. (3), since no sizable loss of proteins out of the gel-membrane system was to be expected in these experiments. A second membrane positioned below the first blotting membrane did not contain any radioactivity after blotting. From this result it cannot be excluded that a part of the protein migrated to the anode. But calculating Be with Eq. (2) did not result in lower blotting eflkiencies, as would have been expected if a significant part of the protein had migrated to the anode. Therefore, the more consistent Bevaluescalculated with Eq. (3) were used (Fig. la). The results show that after 3h almost all blottable protein had bound to the membrane except BSA, the protein with the highest molecular weight among the proteins compared. The blotting efficiency of BSA increased further between 3-5 h. For the other proteins the blotting efficiency could not be increased further by increasing the blotting time to more than 3 h. Standard deviations of optical densities for the spots of the five marker proteins on the membranes after blotting and on the gels before blotting from 6 experiments were in the range of 10 %. The slow decrease of blotting efficiency for CA, TI and P-LG is therefore not significant. For the diagrams shown in Fig. lb-ld, Eq. (2) was used to calculate Be except for BSA. The prob(abi1ity that proteins migrate through the membrane is low if their molecular weight is above 60 000. For BSA we could not detect any protein on a second consecutive Glassybond membrane in addition to that in direct contact with the gel. This was tested under optimum conditions, even in the case where the cathodic blotting buffer contained SDS. For BSA theB,values determined with Eq. (2) were in all cases higher than Bevalues calculated with Eq. (3). Therefore, the more consistent B, values determined with Eq. (3) were preferred for BSA. For the other proteins, where, except for the dependency ofB,on time, a migration through the membrane was observed in some cases, Eq. (2) had to be used. The results obtained when the current was varied between 0.5 and 3.0 mA/cm2 are shown in Fig. lb. For the higher molecular weight proteins CA (29 000) and BSA (66 300) the blotting efficiency increased with increasing current, while the lower molecular weight proteins TI (20 100) and P-LG (1 8 400) showed optimal blotting conditions with 1 mA/cm2, clearly decreasing with 3 mA/cm2. RNase (1 3 600) blotting efficiency decreased slightly when the current was increased from0.5 to 3.0 mA/cm2. With Eq. (4) the percentage of protein remaining in the gel P, was calculated. For example: for P-LG the P, values for 0.5,1, and 3 mA/cm2 were 38.8, 17.3, and 6.2 %, respectively,whereas the percentages of the proteins which bound to the membrane (Be)were 62.3,97.4, and 33.4, respectively. Using 0.5 and 1.0 mA/cm2 the sum of P , and B, results in values of about 100 %, showing that no protein had migrated through the membrane. Using 3 mA/cm2 the sum is 39.6 %, i. e. clearly below 100 %, showing that more than 60 % of the protein migrated through the membrane without binding. In the case of the TI, 26 % of the protein migrated through the membrane. For CA, BSA and RNase the sum of Beand P , was slightly above 100 %, suggesting that no part of the protein was lost by migration through the membrane. The dependency of the blotting efficiency on the ionic strength of the blotting buffer is shown in Fig. lc. Generally, blotting efficiency could be increased by increasing the borate con-
centration. However, in the range of 5 - 10 mM borate the blotting efficiency of the higher molecular weight proteins C A and, especially, BSA was considerably higher than for the lower molecular wei'ght proteins P-LG and TI, i. e. with higher molecular weight proteins, high blotting efficiencies can also be reached at low levels of ionic strength (10 mM borate). RNase already showed a blotting efficiencyof 57.8 % a t 5 m M borate, but did not increase drastically when the ionic strength was increased. At 5 mM borate the sum of Be and P , was 72.0 % for BSA, 27.4 %for CA, 73.9 YOfor TI, 47.2 %for pL G and 87.4 % for RNase, showing that the proteins, especially 0-LG and CA, bound poorly. This is the only case where BSA migrated through the membrane and therefore the blotting efficiency was calculated with Eq. (2). The sum of Be and P, at 10 mM borate was below 100 % for TI, P-LG and RNase with 5 1.3,56.9 and 76.1 %, respectively. With higher ionic strength no protein migrated through the membrane. This was found for all proteins tested. Methanol was added to the blotting buffer to improve binding of the proteins on the membrane. The effect of methanol in different concentrations is shown in Fig. Id. With the exception Table 1. Blotting efficiency of '2sJ-labeled proteins from mouse brain blotted onto Glassybond membranes Optical density blotted gel SGF
Gel
2 1.60") 2.56*) 24.54*) 5.52a) 9.99a) 26.57")
3.26 -
3.17 -
4.47b) 1.89b) 2.87bl 20.36b) 8.25b'
2.93 4.45
3 1 .5OC1 14.13") 2.78'1 18.42c) 7.32') 4.5 IL1 68.71') 2. 14c)
2.48 __ 4.80 1.87 15.03 -
4.27d) 32.25") 5.0Sd) 84.1 7d) 58.9 2d) 7.76d1 5.09d) 70.06d) 12.8Id) 60.93d)
a) b) c) d) e)
-
-
__ 6.79 -
2.90 -
24.26 1.57 23.83 5.29 5.26 14.71 2.59 1.53 2.14 7.05 2.42 8.24 11.55 3.14 8.38 7.59 3.71 41.02 1.85 3.80 29.79 3.19 32.15 25.74 2.72 5.41 23.89 5.95 15.24
P2)
("/.I
B,C) (%)
15.1
112.3 61.3 97.1 95.8 31.7 52.6 55.3 mean value 79.1 __ 57.9 81.0 95.5 14.4 34.6 53.9 29.3 mean value 59.7 26.2 17.6 81.7 112.9 26.1 45.5 25.6 103.7 82.3 21.9 59.7 __ 86.4 mean value 74.8 89.0 21.1 92.4 62.8 41.8 43.7 35.1 106.3 4.1 34.1 46.4 25.0 mean value 57.7 -
High molecular weight/acidic range High molecular weight/neutral range Low molecular weight/acidic range Low molecular weightheutral range Proteins were blotted under optimized blotting conditions: 50 mM borate, pH 9.0,20 %methanol, blotting time 3 h, and 1 mA/cm2 onto Glassybond membranes. The blotting efficiency B, and the percentage of the protein remaining in the gel Pgwere calcultated according to Eqs. (2) and (4). respectively.
Ekctrophoresis 1990,II, 581-588
of BSA, increasing the methanol concentration up to 20 % increased the blotting efficiency. While this effect was quite obvious for the low molecular weight proteins (for example P-LG: without methanol the blotting efficiency was 1.9 %, with 20 % methanol it was 97.4 %), the blotting efficiency increased to a lower degree for CA (43.9 % without methanol, up to 86.9 % with 20 % methanol), and it remained at about 94 %,independently of the methanol concentrationin the case of BSA. The sum of Be and P , without methanol for TI, P-LG and RNase was 33.9, 12.8, and 31.8 96,respectively, clearly showing that most of the protein migrated through the membrane. For BSA the sum was about 100% for all methanol concentrations, showing that no part of the protein migrated through the membrane. For CA the sum was 90.2 % without methanol, showing that perhaps a small amount of protein had migrated through the membrane. With 10 % methanol in the blotting buffer, only low molecular weight proteins migrated through the membrane. The sum of Be and P ,
B 0
585
Blotting efficiency of proteins o n hydrophobic membranes
CX
was 79.1,41.5, and 38.8 %for TI, P-LG and RNase, respectively. In the case of 20 %methanol the sum was about 100 % for all proteins, showing that none of the protein migrated through the membrane. This result could be confirmed by the observation that no protein could be detected on a second Glassybond membrane, laid beneath the first one during blotting. A discontinuous buffer system with 5 % methanol at the cathodic side and 20 %methanol at the anodic side was tested with the aim of increasing the transfer under optimal binding characteristics. However, the blotting efficiencies for the marker proteins could not be improved under these conditions (Fig. Id). As shown in Fig. le, increasing the pH from 8 to 10resulted in optimal blotting efficiencies at pH 9 for TI, P-LG, and RNase, whereas the blotting efficiency for CA increased further when the p H was increased up to 10. The blotting efficiency of BSA did not change by varying the p H between 8 and 10. Protein
2
?
D
Figure 2. Protein patterns and maps obtained with different membranes. ''C-Proteins from mouse embryos were blotted onto (A)Glassybond and (B)Immobilon membrane. Proteins were detected by autoradiography. Calibration was performed with marker proteins as described I 141. The protein maps of (C) Glassybond and (D) Immobilon membrane were obtained by automatic evaluation. The spots selected for membrane comparison are numbered in (C). The numbers correspond to the numbers in Fig. 3.
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P. Jungblut el al.
migrated through the membrane only at pH 8. This was found for CA, TI, P-LG and RNase by considering thesumofB,and P,to be 28.9,75.7,8 1.2, and 95.9 %,respectively. BSAdidnot migrate through the membrane at any pH tested. To continuously support the proteins with SDS during the blotting procedure, SDS was added to the cathodal buffer in concentrations ofO. 1 and 0.5 %. TheeffectofSDS on the blotting efficiency is demonstrated in Fig. If. An improved blotting efficiency in the presence of SDS was only found for CA. For the low molecular weight proteins TI, P-LG and RNase, the transfer out of the gel was complete in the presence of SDS: no protein could be detected in the gels after blotting. However, the ability of the proteins to bind to the membrane was reduced, i.e. the proteins migrated through the membrane. The blotting efficiency of BSA was not influenced by the addition of SDS to the cathodal buffer. Generally, the optimum conditions obtained by the preceding visual investigation were confirmed by the quantitative investigation.
3.2 Blotting efficiency of mouse brain proteins Investigating the blotting parameters for five marker proteins with different molecular weights and isoelectric points, we obtained blotting conditions in which blotting efficiencies in the range of 70-100 % were reached. We used the optimum conditions to test the blotting efficiency for the Glassybond membrane of a complex mixture of natural proteins, i. e. of an extract of mouse brain proteins. All protein spots detectable on the gel were also found on the Glassybond membrane after blotting. From 80 spots detected on the gel, only 20 were still detectable as residual proteins on the gel after blotting. The optical densities of these residual spots were low. The optical densities of 29 proteins from different regions of the pattern are shown in Table 1 under different conditions: on the gel before blotting, on the gel after blotting and on the Glassybond
membrane. The blotting efficiencies calculated with Eq. (2) varied between 25 and 100 % with an overall mean value of 67.8 %.From the dataofTable 1 noinfluenceofthemolecular weight on the blotting efficiency is recognizable, whereas a slight effect of p l can be found. The mean value of Be in the acidic range was 77 % and in the neutral range, 58.7 %.
3.3 Comparison of hydrophobic membranes The optimum blotting conditions found for the Glassybond membrane were used to compare hydrophobic membranes; they were Glassybond, Immobilon, ProBlott, and Selex-20. 14C-Proteinsfrom mouse embryos were separated by 2-DE and blotted onto the four different membranes. The four separation and blotting experiments were run in parallel to increase reproducibility, and were repeated once. The protein pattern of one of the Glassybond membranes and of one ofthe Immobilon membranes as well as the corresponding patterns resulting from the automatic evaluation are shown in Fig. 2. The protein patterns on the membranes consistedof about 450 spots. The patterns were divided into four regions: acididhigh molecular weight, and neutral/low molecular weight. Five spots were selected from each area and quantified. As shown in Fig. 2, only well-resolved spots were taken to avoid errors which may be introduced by our automatic evaluation system. Furthermore, none of the spots selected revealed intensities reaching the saturation level of the blackness of the film. The comparison of the membranes was based on the absolute optical densities of the twenty spots selected. The results of membrane comparison (Fig. 3) can be summarized as follows: (i) While for a specific protein tested some of the membranes may be preferable, none of the membranes displayed an overall superiority with regard to all the proteins tested. (ii) The ProBlott membrane showed a tendency for higher optical densities in all regions. (iii) In the low molecular weight range the spots of the Glassybond membranes had optical densities slightly lower than the other membranes.
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Figure 3. Comparison of different membranes. '"C-labeled mouse proteins were separated by 2-DE and blotted onto the following membranes: Glassybond (triangles), Immobilon (open circles), Selex-20 (squares), and Problott (closed circles). Proteins were detected on the membranes with autoradiography. The optical densities of twenty protein spots from four regions of the protein pattern (a: highmolecular weight/acidic range, b: high molecular weight/neutral range, c: low molecular weight/acidic range and d: low molecular weigbtheutral range) were determined twice for each membrane and each protein. Both single values and mean values are shown.
Eleclrophoresis 1990.11, 58 1-588
Blotting efficiency of proteins on hydrophobic membranes
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4 Discussion Blotting efficiency was studied with the aim offinding blotting conditions by which amaximum amount of proteins of a 2-DE pattern could be transferred from a 2-DE gel to a hydrophobic membrane. Variations in the blotting conditions to improve the transfer (increasing the current/area) resulted in higher blotting efficiencies for higher molecular weight proteins, whereas the blotting efficiency of lower molecular weight proteins decreased. Conditions to improve protein binding on the membrane (increasing the ionic strength, methanol concentration and decreasing the SDS concentration in the blotting buffer), resulted in higher blotting efficiencies for low molecular weight proteins without a decrease of blotting efficiency for higher molecular weight proteins. Using 50 mM borate, pH 9.0, and 20 % methanol as blotting buffer and a current of 1 mA/cm2, with a blotting time of 3 h, high blotting efficiencies can be achieved for many proteins of complex mixtures separated by 2-DE with a 15 % acrylamide gel in the second dimension. For single proteins, the blotting conditions used here may serve as a good start for optimization experiments. The results shown, as well as observations from studies on one-dimensional gels 161, imply a blotting mechanism as illustrated in Fig. 4. After 2-DE the protein in the gel is loaded with SDS and, therefore, has a negative charge. During blotting, the protein-SDS complexes migrate towards the anode in a medium of continuously decreasing concentration of free SDS. As shown by Putnam and Neurath [ 161,in different protein-SDS complexes, binding of the detergent depends on the SDS-to-protein ratio. Therefore, a decreased SDS concentration will reduce the SDS concentration on the protein surface, disclosing hydrophobic sites which may interact with the hydrophobic membrane. Low molecular weight proteins bind to a lower degree because the protein-SDS complexes migrate too fast, so that the amount of SDS in these complexes is not sufficiently decreased. The high transfer velocity may result from an excessively high current. The decreased protein binding to the membrane may be additionally caused by conditions weakening hydrophobic interactions as, for example, low ionic strength and low methanol concentration. Our results corroborate that at high current/area levels, low ionic strength, low methanol and/or high SDS concentration in the blotting buffer, substantial amounts of low molecular weight proteins migrate through the membrane. As aresult ofthedissociationofSDS, migration oftheproteinSDS complexes will slow down and some of the proteins, especially high-molecular weight proteins, may even become insoluble and precipitate in the gel. Increasing the blotting time above 3 h did not increase blotting efficiency (Fig. la), not because of protein loss by migration through the membrane but rather by residual protein immobilized in the gel. This was also found for proteins blotted from one-dimensional gels for more than 5h where the residual protein had lost all ofits SDS and, consequently, could not be transferred. The addition of SDS to the cathodal blotting buffer led to 100 % blotting efficiency for CA. The blotting behavior of a protein with known molecular weight cannot be predicted because the amino acid composition and sequence surely have an additional influence on the blotting efficiency. However, a better understanding of the blotting mechanism may make it possible to improve the blotting conditions more systematically.
3 cathode
filter
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anode
Figure4. Postulatedblottingmechanism. (a)IntheSDSgeltheprotein(P)is loaded with SDS (dash in small circles). (b) The protein-SDS complexes migrate out of the gel onto the membrane. Because of the decreasing concentration of SDS in the surroundings of the protein-SDS complexes, SDS is also lost from the protein during this migration. (c) SDS is stripped off from the protein and migrates towards the anode, whereas the protein remains bound on the membrane.
According to our experience, two major problems may occur: (i) High molecular weight proteins tend to be retained in the gel and for improved blotting efficiency the current should be increased, the methanol concentration decreased and SDS may be used in the cathodal blotting buffer along with a prolonged blotting time. (ii) In the case of low molecular weight proteins which do not bind adequately onto the membrane and migrate to the cathode, the current should be decreased and simultaneously the ionic strength as well as methanol concentration increased to improve binding of these proteins on the membrane. Another important parameter for the blotting efficiency is the hydrophobicity of the membrane used. Among the four membranes tested, namely Glassybond, Immobilon, ProBlott and Selex-20, listed according to increasing hydrophobicity, the ProBlott membrane showed slightly higher protein binding, but not for all single proteins. Glassybond membranes, characterized by a lower hydrophobicity, showed a lower binding capacity for low molecular weight proteins. Blotting conditions for an individual protein can be optimized by either varying the conditions as described above or by selecting an appropriate membrane. For sequencing, however, not only
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the blottingefficiency, but alsothestability ofthemembrane in the sequenator should be considered. Under these conditions, for two of the proteins tested the Glassybond membrane membranes produced higher initial yields than the as described by Eckerskorn and Lottspeich [ 171.
Schaubitzerfor exThe authors are grateful to Mrs. cellent technical assistance, to Dr. Jean iVowakf o r automatic evaluation of protein patterns, and to Dr. Klaus Hinsch (Institute ofPharmacology, Berlin, FRG)for the iodination of marker proteins and mouse brain proteins. Received March 15, 1990
5 References
Electrophoresis 1990,11,581-588
15 I Matsudaira, P., J . B i d . Chem. 1987,262,10035-10038. [61 Eckerskorn, C., Mewes, W., Goretzki, H. and Lottspeich, F., Eur. J .
Biochem. 1988,176,509-519. [71 Eckerskorn, C . ,J u n g b U P.,Mewes,W., Klose, J. and Lottspeich, F., Electrophoresis 1988,9,830-838. [Sl Walsh, M., MeDougall, J . and Wittmann-Liebold, B.,Biochemistry 1988,27,6867-6876. 191 Aebersold, R.H., Leavitt, J., Saavedra, R. A., Hood, L. E. and Kent, S. B. H., Proc. Natl. Acad. Sci. USA 1987,84,6970-6974. [I01 Eckerskorn, C. and Lottspeich, F., Chromatographia 1989, 28, 92-94. 11 1 I Peterson, G. L., Anal. Biochem. 1977,83,346-356. I121 Salacinski, P. R. P., McLean, C., Sykes, J . E. C.,Clement-Jones,V. V. and Lowry, P. J., Anal. Biochem.l981,117,136-146. I131 Klose, J., in: Tschesche, H. (Ed.), Modern Merhods in Protein ChemisrryReview Articles, Walter de Gruyter Verlag, Berlin 1983, pp. 49-78. I141 Jungblut, P. and Seifert, R., J. Biochem. Biophys. Methods 1990,in
I l l OFarrell, P. H., J. Biol. Chem. 1975,250,4007-4021. 121 Klose, J., Humangenetik 1975,26,23 1-243. I31 Vandekerckhove, J., Bauw, G., Puype, M., Van Damme, J. and Van
press. 1151 Prehm, J., Jungblut, P. and Klose, J., Elecfrophoresis 1987, 8 ,
Montagu, M., Eur. J. Biochem. 1985,152,9-19. 141 Aebersold, R., Teplow, D., Hood, L. E. arid Kent, S. B. H., J . Biol. Chem. 1986,261,4229-4238.
[I61 Putnam,F. W.andNeurath,H.,J.Biol. Chem. 1945,159,195-209. I171 Eckerskorn, C. and Lottspeich, F., J . Proi. Chem. 1990,in press.
562-572.
Peter Jungblut Christoph Eckerskorn2 Friedrich Lottspeich2 Joachim Klose 'Institut fiir Humangenetik,Institut fur Toxikologie und Embryonalpharmakologie,Freie Universitat Berlin 2Max-Planck-Institutfur Biochemie, Genzentrum, Martinsried
Blotting efficiency of proteins on hydrophobic membranes
58 1
Blotting efficiency investigated by using two-dimensional electrophoresis, hydrophobic membranes and proteins from different sources Purification and chemical characterization of proteins may be achieved by combining two-dimensional electrophoresis (2-DE) and microsequencing or amino acid analysis. To enable this combination, the protein has to be transferred as completely as possible from the gel into the sequencer. In this study hydrophobic membranes were used as support for the transfer and proteins were transferred from the gels onto the membranes by semidry blotting. Blotting conditions were optimized to obtain high blotting efficiencies for as many proteins of a complex 2-DE pattern as possible. Under optimized conditions, blotting efficiencies between 60 % and 100 % were obtained for five marker proteins; the mean values from four regions of a 2-DE pattern from 29 unknown proteins of a complex protein mixture from mouse brain were between 60 % and 79 %. The four commercially available hydrophobic membranes that were compared showed only slight differences in protein amount on the membranes after blotting for whole protein patterns, whereas single proteins occurred with higher amounts on either one or the other membrane. The results ofthe blotting optimization allowed us to suggest a blotting mechanism with which systematic improvement of the blotting conditions is possible for problematic proteins.
1 Introduction Two-dimensional gel electrophoresis (2-DE) using the combination of isoelectric focusing (IEF) and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis [ I , 21 is the method with the highest resolution in protein analysis. At present, N-terminal sequencing methods are sensitive enough to sequence pmole amounts of proteins. With the development of membranes which can immobilize proteins blotted from polyacrylamide gels and that are directly applicable to the sequencing machine, 2-DE and microsequencing could be combined. The use ofone and the same support for blotting as well as for sequencing prevents loss and chemical modifications of the protein. Vandekerckhove et al. 131 and Aebersold et al. [41 developed positively charged membranes, while Matsudaira (51 and Eckerskorn et al. 161 introduced hydrophobic membranes for the immobilization of the proteins. The two main advantages of hydrophobic membranes are higher protein binding capacities and easy detection of proteins with commercially available protein dyes. Positively charged as well as hydrophobic membranes were used for blotting and sequencing ofproteinsfrom 2-DEgels [3-5,7,81, and anumber of proteins in the 2-DE patterns were identified by sequence comparison with the help of a sequence data base (71. To be able to also sequence proteins which occur in low amounts on the membrane after Coomassie Brilliant Blue staining, or which require the analysis ofinternal sequences [9,101, ahigh blotting efficiency is a prerequisite. In this investigation an attempt was made to find optimum conditions for blotting complex 2-DE protein patterns using
Correspondence: Dr. Peter Jungblut, Institut fur Toxikologie und Embryonalpharmakologie, Freie Universitat Berlin, Garystr. 5 , D-1000 Berlin 3 3 , Federal Republic of Germany Abbreviations: 2-DE, two-dimensional gel electrophoresis; f3-LG,bovine P-lactoglobulin; Be, blotting efficiency; BSA, bovine serum albumin; CA bovine carbonic anhydrase; IEF, isoelectric focusing;P,, protein remaining in the gel during blotting, RNase, bovine ribonuclease; SDS, sodium dodecyl sulfate; TI, soybean trypsin inhibitor
0VCH Verlagsgesellschaft mbH D-6940 Weinheim, 1990
the semidry blotting procedure. Compared to the conventional vertical blots in buffer tanks, semidry blotting has the advantages of reduced buffer consumption, minimized reactive impurities, reduced risk of formation of air bubbles due to a straightforward assembly, and reduced transfer time and heat development. On the basis of the results, a blotting mechanism for semidry blotting was suggested which could improve the blotting efficiency. Automatic evaluation ofthe 2D E patterns was used to compare different commercially available hydrophobic membranes.
2 Materials and methods 2.1 Protein sample preparation Solubilized proteins were extracted from the brains and embryos of mice, strain DBA/2J. Four brains were washed with 0.9 % NaCl to remove blood. After freezing and thawing, the brains were homogenized (five up-and-down strokes) in a 5 mL homogenizer in one volume of distilled water containing protease inhibitors. The final concentrations of the inhibitors were 5 mM ethylenediaminetetraacetate (EDTA), I mM phenylmethylsulfonyl fluoride (PMSF) and 0.1 pg pepstatin A (all from Sigma, Taufkirchen, FRG). The homogenate was placed into six tubes and centrifuged in a Beckman TI-50 rotor for 40 min at 225 000 g. The supernatants were combined, vortexed, and frozen in 200 pL aliquots. Each of the ten aliquots obtained contained 7.1 mg/mL protein as determined by the method of Peterson [ 1I]. A portion ofthe brain proteins was iodinated by the method described by Salacinski et al. [ 121. Twenty pL of the protein preparation were incubated for 15 min together with a chloramine T-bead (Iodobeads, Pierce, Oud-Beijerland, The Netherlands) and with NalZ5J,the radioactivity of which was 0.4 mCi. The iodinated proteins were separated from the free 25J-bygel filtration on aSephadex G25 (Pharmacia-LKB, Uppsala, Sweden) column. Marker proteins were also used in this study and iodinated as well. The marker protein mixture contained 2 pg each of bovine serum albumin (BSA, Sigma), bovine P-lactoglobulin (P-LG, Sigma), bovine ribonuclease (RNase, Sigma), bovine carbonic 0173-0835/90/0707-058 I $3.50+.25/0
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anhydrase (CA, Serva, Heidelberg, F R G ) and soybean trypsin inhibitor (TI, Sigma), solubilized together in 20 pL of a buffer containing 4 mM KH,PO,, 10 mM Na,HPO, and 120 mM NaC1. The marker proteins RNase, P-LG, TI, C A and BSA have the following molecular weights and p l values: 13 600, 7.8; 18 400,5.3; 20 100,4.5; 29 000,5.91;and 66 300,4.8, respectively, The mixture (20 pL) was iodinated with 0.25 mCi NaI2'J as described above. The radioactiivity of the '25J-labeled marker proteins obtained was 77 000 cpm/pL, that of the labeled brain proteins 200 000 cpm/pL. Solubilized proteins from mouse embryos were prepared and labeled with I4Camino acids as described by Klose [ 131. IJrea (Bio-Rad, Richmond, CA, USA), mercaptoethanol (Merck, Darmstadt, FRG) and Servalyt carrier ampholytes p H 5-7 (Serva) were added to all the protein preparations to obtain final concentrations of 9 M, 5 % v/v and 2 % v/v, respectively. The resulting solutions were used as protein samples for 2-DE.
2.2 2-DE The 2-DE technique first described by Klose [ 21 and modified as described by Jungblut and Seifert [ 141 was used to separate the proteins. The radioactivity ofthe sample applied to the IEF gels was determined and amounted to 801 000 cpm for marker proteins, 400 000 cprn for brain proteins and 125 000 cpm for the embryo proteins. Parameters influencing the blotting efficiency are the acrylamide concentration (the porosity) of the SDS gel and the ionic strength in that gel after 2-DE. The acrylamide concentration was 15 % w/v acrylamide, 0.2 % w/v N,N'-methylenebisacrylamide(1 5.2 % T, 1.3 % C). The ionic strength in the gel after 2-DE shoulld correspond to that ofthe electrode buffer,which was 25 mM Tris, 192 mMglycine and 0.1 % w/v SDS. The size of the slab gels was 6.5 (running direction) x 8.5 x 0.15 cm. To prevent loss of proteins by diffusion, the SDS gels were not equilibrateld with blotting buffer but were directly subjected to the blotting procedure.
2.3 Electroblotting Electroblotting was performed by the semidry procedure using a Sartoblot I1 blotting apparatus (Slartorius, Gottingen, FRG) with graphite electrodes. In a preceding investigation mouse brain proteins were blotted onto the siliconized glass fiber membrane Glassybond (Biometra, Gottingen, F R G ) or onto the polyvinylidene difluoride membrane Immobilon (Millipore, Bedford, MA, USA). The proteins were detected with Coomassie Brilliant Blue R-250 as described 171. The blotting parameters, i. e. the composition (ionic strength, pH, methanol and SDS content) of the blotting buffer. blotting time and current/cm2, were varied. The protein patterns obtained on the membranes were examinedl visually with regard to completeness in spot composition and with regard to spot intensities. With this more qualitative approach higher intensities of protein spots on the membrane were obtained with a blotting buffer containing 50 mM borate, p H 9.0, and 20 % methanol by blotting with 1 mA/cm2 for 3 h. The question of whether these conditions were optimal for the blotting efficiency was tested by quantitative deteirmination ofthe blotting efficiency, which was performed with the Glassybond membrane. The membrane was washed for about 30 s in acetone and then equilibrated in blotting buffer for 20 min with three changes. The blotting buffer contained borate and methanol in different concentrations witlh varying pH values.
Electrophoresis 1990, 11, 581-588
The effect of different SDS concentrations in the cathodic blotting buffer was also investigated. Filter papers (Nr. 2668/ ~ 2 4 9 3 4 Schleicher , and Schuell, Dassel, FRG) were washed for 30 rnin with three changes. The graphite electrodes were soaked with distilled water before setting up the blotting sandwich. The lower electrode was used as anode and the sandwich was built up from the anodic side as follows: the membrane waslaidonto threelayersoffi1terpaper:thegelwas transferred onto the membrane and overlaid with three layers of filter paper. Air bubbles between the layers were removed by gently rolling a glass rod over each layer but not over the membrane. Electrotransfer was performed at different levels of constant current. Blotting time was varied between 0.5 and 5 h. For the comparison of hydrophobic membranes, four membranes were used: Glassybond, Immobilon, another polyvinylidene difluoride membrane ProBlott (Applied Biosystems, Foster City, CA, USA) and a polypropylene membrane Selex-20 (Schleicher and Schuell, Dassel, FRG). Glassybond was prewetted in acetone and washed with blotting buffer as described above. Immobilon was washed for 10 rnin in methanol and then equilibrated in blotting buffer for 20 min with three changes. Selex-20 and ProBlott membranes were washed with methanol for 10 rnin and then used directly to set up the blotting sandwich as described for the Glassybond and Immobilon membrane.
2.4 Detection of proteins on membranes and gels Radioactively labeled proteins were detected by autoradiography. The gels were dried before autoradiography as described by Klose 131. The membranes were air-dried at room temperature for a few hours, Dried gels or membranes were laid on Kodak X-Omat A R 5 X-ray film and exposed for 2-5 days.
2.5 Automatic evaluation For quantitative investigations of protein patterns an automatic evaluation that included a video camera and a computer was performed as described by Prehm et al. [ 151. Optical densities were measured and care was taken that none ofthe spots selected had an intensity reaching the saturation level of the blackness of the film.
2.6 Determination of blotting efficiency To confirm the results of our qualitative investigation of the blotting conditions we developed a quantitative test which defines blotting efficiency by the equation
B , is the blotting efficiency, mmembrane is the amount of protein on the membrane after blotting and ingelis the amount of protein in the gel before blotting. The amount of protein in the gel cannot be determined directly, but both mgeiand mmembranemaY be determined indirectly by measuring the optical densities obtained by the use of radioactively labeled proteins and autoradiography. In this way the blotting efficiency can be determined directly from the measured optical densities:
Blotting efficiency of proteins on hydrophobic membranes
Electrophoresis 1990,II, 581-588
ODmembrane is the optical density of the protein investigated in the membrane and ODgelisthe optical density oftheproteininvestigated in the gel before blotting. When using Eq. (2) one has to consider the possibility that the radioactivity may be quenched differently by dried gels and membranes. No quenching effects occur with lZ5J-labeledproteins, and, therefore, '25J-labeled marker proteins were used to quantify the blotting efficiency under different blotting conditions. For the determination of ODgelit is not possible to use the same gel from which the proteins are blotted onto the membrane. Therefore, pattern comparison has to be performed on parallel gels. To minimize differences between the two parallel gels due to problems in reproducibility of 2-DE, the ODgeIvalues were calculated as a mean value from six gels. Another way to determine Beis to measure the O D of the proteins possibly left on the gel after blotting (ODblotged,and to set the sum of ODblotgeland ODmembraneequal 100 %. Beisthencalculated by
583
thus leading to more precise results. Therefore, if protein loss during the blotting procedure can be excluded, Eq. (3) should be used. If a blotting efficiency below 100 %was obtained, the question was whether the protein was not transferred from the gel, or whether the protein has passed the membrane. In this case, ODblotgel was determined and the percentage of the protein left in the gel P, was calculated by Eq. (4).
P,=
ODbIotgel X 100
(4)
ODgeI For OD,,I a mean value of six analyses was used.
3 Results 3.1 Blotting eaciency of marker proteins
(3) In this case it is assumed that no protein was lost during the blotting procedure. The calculation of Bewith Eq. (3) has the advantage that no OD-values of parallel gels have to be used,
1
2
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In Fig. 1 the effect of varying blotting time, current/area, ionic strength, methanol concentration, pH, and SDS concentration on the blotting efficiency for Glassybond membranes is shown. The blotting efficiency of marker proteins was determined by quantitative analysis. The optimum conditions ob-
10
0
0.5
0.1
505-concmtration I%l Icathdd huffcrl
5u)hde
ZOrncdC
Figure 1. Blotting efficiency of '25J-labeled marker proteins. RNase (closed squares), trypsin inhibitor (open squares), P-LG (triangles), CA (open circles), and BSA (closed circles) were separated by 2-DE and blotted onto Glassybond membranes. The blotting buffer consisted of 50 mM borate, pH 9.0,20 %methanol. Proteins were blotted with 1 mA/cm2 for 3 h. One of these parameters was varied in each of the six diagrams (a)-(0. Proteins were detected by autoradiography. Blotting efficiency was determined in (a) and for BSA (all diagrams except c, 5 mM with Eq. (3). For the other proteinsindiagram (b)-(0Eq. (2)was used.
584
Eleclrophoresis 1990, 11, 5XI-5XX
P. Jungblut el a!.
tained by the preceding qualitative investigation as described in Section 2.3 were used. Only one parameter was varied in each investigation. The blotting efficiency depends on the blotting time (Fig. la) and was calculated according to Eq. (3), since no sizable loss of proteins out of the gel-membrane system was to be expected in these experiments. A second membrane positioned below the first blotting membrane did not contain any radioactivity after blotting. From this result it cannot be excluded that a part of the protein migrated to the anode. But calculating Be with Eq. (2) did not result in lower blotting eflkiencies, as would have been expected if a significant part of the protein had migrated to the anode. Therefore, the more consistent Bevaluescalculated with Eq. (3) were used (Fig. la). The results show that after 3h almost all blottable protein had bound to the membrane except BSA, the protein with the highest molecular weight among the proteins compared. The blotting efficiency of BSA increased further between 3-5 h. For the other proteins the blotting efficiency could not be increased further by increasing the blotting time to more than 3 h. Standard deviations of optical densities for the spots of the five marker proteins on the membranes after blotting and on the gels before blotting from 6 experiments were in the range of 10 %. The slow decrease of blotting efficiency for CA, TI and P-LG is therefore not significant. For the diagrams shown in Fig. lb-ld, Eq. (2) was used to calculate Be except for BSA. The prob(abi1ity that proteins migrate through the membrane is low if their molecular weight is above 60 000. For BSA we could not detect any protein on a second consecutive Glassybond membrane in addition to that in direct contact with the gel. This was tested under optimum conditions, even in the case where the cathodic blotting buffer contained SDS. For BSA theB,values determined with Eq. (2) were in all cases higher than Bevalues calculated with Eq. (3). Therefore, the more consistent B, values determined with Eq. (3) were preferred for BSA. For the other proteins, where, except for the dependency ofB,on time, a migration through the membrane was observed in some cases, Eq. (2) had to be used. The results obtained when the current was varied between 0.5 and 3.0 mA/cm2 are shown in Fig. lb. For the higher molecular weight proteins CA (29 000) and BSA (66 300) the blotting efficiency increased with increasing current, while the lower molecular weight proteins TI (20 100) and P-LG (1 8 400) showed optimal blotting conditions with 1 mA/cm2, clearly decreasing with 3 mA/cm2. RNase (1 3 600) blotting efficiency decreased slightly when the current was increased from0.5 to 3.0 mA/cm2. With Eq. (4) the percentage of protein remaining in the gel P, was calculated. For example: for P-LG the P, values for 0.5,1, and 3 mA/cm2 were 38.8, 17.3, and 6.2 %, respectively,whereas the percentages of the proteins which bound to the membrane (Be)were 62.3,97.4, and 33.4, respectively. Using 0.5 and 1.0 mA/cm2 the sum of P , and B, results in values of about 100 %, showing that no protein had migrated through the membrane. Using 3 mA/cm2 the sum is 39.6 %, i. e. clearly below 100 %, showing that more than 60 % of the protein migrated through the membrane without binding. In the case of the TI, 26 % of the protein migrated through the membrane. For CA, BSA and RNase the sum of Beand P , was slightly above 100 %, suggesting that no part of the protein was lost by migration through the membrane. The dependency of the blotting efficiency on the ionic strength of the blotting buffer is shown in Fig. lc. Generally, blotting efficiency could be increased by increasing the borate con-
centration. However, in the range of 5 - 10 mM borate the blotting efficiency of the higher molecular weight proteins C A and, especially, BSA was considerably higher than for the lower molecular wei'ght proteins P-LG and TI, i. e. with higher molecular weight proteins, high blotting efficiencies can also be reached at low levels of ionic strength (10 mM borate). RNase already showed a blotting efficiencyof 57.8 % a t 5 m M borate, but did not increase drastically when the ionic strength was increased. At 5 mM borate the sum of Be and P , was 72.0 % for BSA, 27.4 %for CA, 73.9 YOfor TI, 47.2 %for pL G and 87.4 % for RNase, showing that the proteins, especially 0-LG and CA, bound poorly. This is the only case where BSA migrated through the membrane and therefore the blotting efficiency was calculated with Eq. (2). The sum of Be and P, at 10 mM borate was below 100 % for TI, P-LG and RNase with 5 1.3,56.9 and 76.1 %, respectively. With higher ionic strength no protein migrated through the membrane. This was found for all proteins tested. Methanol was added to the blotting buffer to improve binding of the proteins on the membrane. The effect of methanol in different concentrations is shown in Fig. Id. With the exception Table 1. Blotting efficiency of '2sJ-labeled proteins from mouse brain blotted onto Glassybond membranes Optical density blotted gel SGF
Gel
2 1.60") 2.56*) 24.54*) 5.52a) 9.99a) 26.57")
3.26 -
3.17 -
4.47b) 1.89b) 2.87bl 20.36b) 8.25b'
2.93 4.45
3 1 .5OC1 14.13") 2.78'1 18.42c) 7.32') 4.5 IL1 68.71') 2. 14c)
2.48 __ 4.80 1.87 15.03 -
4.27d) 32.25") 5.0Sd) 84.1 7d) 58.9 2d) 7.76d1 5.09d) 70.06d) 12.8Id) 60.93d)
a) b) c) d) e)
-
-
__ 6.79 -
2.90 -
24.26 1.57 23.83 5.29 5.26 14.71 2.59 1.53 2.14 7.05 2.42 8.24 11.55 3.14 8.38 7.59 3.71 41.02 1.85 3.80 29.79 3.19 32.15 25.74 2.72 5.41 23.89 5.95 15.24
P2)
("/.I
B,C) (%)
15.1
112.3 61.3 97.1 95.8 31.7 52.6 55.3 mean value 79.1 __ 57.9 81.0 95.5 14.4 34.6 53.9 29.3 mean value 59.7 26.2 17.6 81.7 112.9 26.1 45.5 25.6 103.7 82.3 21.9 59.7 __ 86.4 mean value 74.8 89.0 21.1 92.4 62.8 41.8 43.7 35.1 106.3 4.1 34.1 46.4 25.0 mean value 57.7 -
High molecular weight/acidic range High molecular weight/neutral range Low molecular weight/acidic range Low molecular weightheutral range Proteins were blotted under optimized blotting conditions: 50 mM borate, pH 9.0,20 %methanol, blotting time 3 h, and 1 mA/cm2 onto Glassybond membranes. The blotting efficiency B, and the percentage of the protein remaining in the gel Pgwere calcultated according to Eqs. (2) and (4). respectively.
Ekctrophoresis 1990,II, 581-588
of BSA, increasing the methanol concentration up to 20 % increased the blotting efficiency. While this effect was quite obvious for the low molecular weight proteins (for example P-LG: without methanol the blotting efficiency was 1.9 %, with 20 % methanol it was 97.4 %), the blotting efficiency increased to a lower degree for CA (43.9 % without methanol, up to 86.9 % with 20 % methanol), and it remained at about 94 %,independently of the methanol concentrationin the case of BSA. The sum of Be and P , without methanol for TI, P-LG and RNase was 33.9, 12.8, and 31.8 96,respectively, clearly showing that most of the protein migrated through the membrane. For BSA the sum was about 100% for all methanol concentrations, showing that no part of the protein migrated through the membrane. For CA the sum was 90.2 % without methanol, showing that perhaps a small amount of protein had migrated through the membrane. With 10 % methanol in the blotting buffer, only low molecular weight proteins migrated through the membrane. The sum of Be and P ,
B 0
585
Blotting efficiency of proteins o n hydrophobic membranes
CX
was 79.1,41.5, and 38.8 %for TI, P-LG and RNase, respectively. In the case of 20 %methanol the sum was about 100 % for all proteins, showing that none of the protein migrated through the membrane. This result could be confirmed by the observation that no protein could be detected on a second Glassybond membrane, laid beneath the first one during blotting. A discontinuous buffer system with 5 % methanol at the cathodic side and 20 %methanol at the anodic side was tested with the aim of increasing the transfer under optimal binding characteristics. However, the blotting efficiencies for the marker proteins could not be improved under these conditions (Fig. Id). As shown in Fig. le, increasing the pH from 8 to 10resulted in optimal blotting efficiencies at pH 9 for TI, P-LG, and RNase, whereas the blotting efficiency for CA increased further when the p H was increased up to 10. The blotting efficiency of BSA did not change by varying the p H between 8 and 10. Protein
2
?
D
Figure 2. Protein patterns and maps obtained with different membranes. ''C-Proteins from mouse embryos were blotted onto (A)Glassybond and (B)Immobilon membrane. Proteins were detected by autoradiography. Calibration was performed with marker proteins as described I 141. The protein maps of (C) Glassybond and (D) Immobilon membrane were obtained by automatic evaluation. The spots selected for membrane comparison are numbered in (C). The numbers correspond to the numbers in Fig. 3.
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P. Jungblut el al.
migrated through the membrane only at pH 8. This was found for CA, TI, P-LG and RNase by considering thesumofB,and P,to be 28.9,75.7,8 1.2, and 95.9 %,respectively. BSAdidnot migrate through the membrane at any pH tested. To continuously support the proteins with SDS during the blotting procedure, SDS was added to the cathodal buffer in concentrations ofO. 1 and 0.5 %. TheeffectofSDS on the blotting efficiency is demonstrated in Fig. If. An improved blotting efficiency in the presence of SDS was only found for CA. For the low molecular weight proteins TI, P-LG and RNase, the transfer out of the gel was complete in the presence of SDS: no protein could be detected in the gels after blotting. However, the ability of the proteins to bind to the membrane was reduced, i.e. the proteins migrated through the membrane. The blotting efficiency of BSA was not influenced by the addition of SDS to the cathodal buffer. Generally, the optimum conditions obtained by the preceding visual investigation were confirmed by the quantitative investigation.
3.2 Blotting efficiency of mouse brain proteins Investigating the blotting parameters for five marker proteins with different molecular weights and isoelectric points, we obtained blotting conditions in which blotting efficiencies in the range of 70-100 % were reached. We used the optimum conditions to test the blotting efficiency for the Glassybond membrane of a complex mixture of natural proteins, i. e. of an extract of mouse brain proteins. All protein spots detectable on the gel were also found on the Glassybond membrane after blotting. From 80 spots detected on the gel, only 20 were still detectable as residual proteins on the gel after blotting. The optical densities of these residual spots were low. The optical densities of 29 proteins from different regions of the pattern are shown in Table 1 under different conditions: on the gel before blotting, on the gel after blotting and on the Glassybond
membrane. The blotting efficiencies calculated with Eq. (2) varied between 25 and 100 % with an overall mean value of 67.8 %.From the dataofTable 1 noinfluenceofthemolecular weight on the blotting efficiency is recognizable, whereas a slight effect of p l can be found. The mean value of Be in the acidic range was 77 % and in the neutral range, 58.7 %.
3.3 Comparison of hydrophobic membranes The optimum blotting conditions found for the Glassybond membrane were used to compare hydrophobic membranes; they were Glassybond, Immobilon, ProBlott, and Selex-20. 14C-Proteinsfrom mouse embryos were separated by 2-DE and blotted onto the four different membranes. The four separation and blotting experiments were run in parallel to increase reproducibility, and were repeated once. The protein pattern of one of the Glassybond membranes and of one ofthe Immobilon membranes as well as the corresponding patterns resulting from the automatic evaluation are shown in Fig. 2. The protein patterns on the membranes consistedof about 450 spots. The patterns were divided into four regions: acididhigh molecular weight, and neutral/low molecular weight. Five spots were selected from each area and quantified. As shown in Fig. 2, only well-resolved spots were taken to avoid errors which may be introduced by our automatic evaluation system. Furthermore, none of the spots selected revealed intensities reaching the saturation level of the blackness of the film. The comparison of the membranes was based on the absolute optical densities of the twenty spots selected. The results of membrane comparison (Fig. 3) can be summarized as follows: (i) While for a specific protein tested some of the membranes may be preferable, none of the membranes displayed an overall superiority with regard to all the proteins tested. (ii) The ProBlott membrane showed a tendency for higher optical densities in all regions. (iii) In the low molecular weight range the spots of the Glassybond membranes had optical densities slightly lower than the other membranes.
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Figure 3. Comparison of different membranes. '"C-labeled mouse proteins were separated by 2-DE and blotted onto the following membranes: Glassybond (triangles), Immobilon (open circles), Selex-20 (squares), and Problott (closed circles). Proteins were detected on the membranes with autoradiography. The optical densities of twenty protein spots from four regions of the protein pattern (a: highmolecular weight/acidic range, b: high molecular weight/neutral range, c: low molecular weight/acidic range and d: low molecular weigbtheutral range) were determined twice for each membrane and each protein. Both single values and mean values are shown.
Eleclrophoresis 1990.11, 58 1-588
Blotting efficiency of proteins on hydrophobic membranes
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4 Discussion Blotting efficiency was studied with the aim offinding blotting conditions by which amaximum amount of proteins of a 2-DE pattern could be transferred from a 2-DE gel to a hydrophobic membrane. Variations in the blotting conditions to improve the transfer (increasing the current/area) resulted in higher blotting efficiencies for higher molecular weight proteins, whereas the blotting efficiency of lower molecular weight proteins decreased. Conditions to improve protein binding on the membrane (increasing the ionic strength, methanol concentration and decreasing the SDS concentration in the blotting buffer), resulted in higher blotting efficiencies for low molecular weight proteins without a decrease of blotting efficiency for higher molecular weight proteins. Using 50 mM borate, pH 9.0, and 20 % methanol as blotting buffer and a current of 1 mA/cm2, with a blotting time of 3 h, high blotting efficiencies can be achieved for many proteins of complex mixtures separated by 2-DE with a 15 % acrylamide gel in the second dimension. For single proteins, the blotting conditions used here may serve as a good start for optimization experiments. The results shown, as well as observations from studies on one-dimensional gels 161, imply a blotting mechanism as illustrated in Fig. 4. After 2-DE the protein in the gel is loaded with SDS and, therefore, has a negative charge. During blotting, the protein-SDS complexes migrate towards the anode in a medium of continuously decreasing concentration of free SDS. As shown by Putnam and Neurath [ 161,in different protein-SDS complexes, binding of the detergent depends on the SDS-to-protein ratio. Therefore, a decreased SDS concentration will reduce the SDS concentration on the protein surface, disclosing hydrophobic sites which may interact with the hydrophobic membrane. Low molecular weight proteins bind to a lower degree because the protein-SDS complexes migrate too fast, so that the amount of SDS in these complexes is not sufficiently decreased. The high transfer velocity may result from an excessively high current. The decreased protein binding to the membrane may be additionally caused by conditions weakening hydrophobic interactions as, for example, low ionic strength and low methanol concentration. Our results corroborate that at high current/area levels, low ionic strength, low methanol and/or high SDS concentration in the blotting buffer, substantial amounts of low molecular weight proteins migrate through the membrane. As aresult ofthedissociationofSDS, migration oftheproteinSDS complexes will slow down and some of the proteins, especially high-molecular weight proteins, may even become insoluble and precipitate in the gel. Increasing the blotting time above 3 h did not increase blotting efficiency (Fig. la), not because of protein loss by migration through the membrane but rather by residual protein immobilized in the gel. This was also found for proteins blotted from one-dimensional gels for more than 5h where the residual protein had lost all ofits SDS and, consequently, could not be transferred. The addition of SDS to the cathodal blotting buffer led to 100 % blotting efficiency for CA. The blotting behavior of a protein with known molecular weight cannot be predicted because the amino acid composition and sequence surely have an additional influence on the blotting efficiency. However, a better understanding of the blotting mechanism may make it possible to improve the blotting conditions more systematically.
3 cathode
filter
gel
membrane filter
anode
Figure4. Postulatedblottingmechanism. (a)IntheSDSgeltheprotein(P)is loaded with SDS (dash in small circles). (b) The protein-SDS complexes migrate out of the gel onto the membrane. Because of the decreasing concentration of SDS in the surroundings of the protein-SDS complexes, SDS is also lost from the protein during this migration. (c) SDS is stripped off from the protein and migrates towards the anode, whereas the protein remains bound on the membrane.
According to our experience, two major problems may occur: (i) High molecular weight proteins tend to be retained in the gel and for improved blotting efficiency the current should be increased, the methanol concentration decreased and SDS may be used in the cathodal blotting buffer along with a prolonged blotting time. (ii) In the case of low molecular weight proteins which do not bind adequately onto the membrane and migrate to the cathode, the current should be decreased and simultaneously the ionic strength as well as methanol concentration increased to improve binding of these proteins on the membrane. Another important parameter for the blotting efficiency is the hydrophobicity of the membrane used. Among the four membranes tested, namely Glassybond, Immobilon, ProBlott and Selex-20, listed according to increasing hydrophobicity, the ProBlott membrane showed slightly higher protein binding, but not for all single proteins. Glassybond membranes, characterized by a lower hydrophobicity, showed a lower binding capacity for low molecular weight proteins. Blotting conditions for an individual protein can be optimized by either varying the conditions as described above or by selecting an appropriate membrane. For sequencing, however, not only
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the blottingefficiency, but alsothestability ofthemembrane in the sequenator should be considered. Under these conditions, for two of the proteins tested the Glassybond membrane membranes produced higher initial yields than the as described by Eckerskorn and Lottspeich [ 171.
Schaubitzerfor exThe authors are grateful to Mrs. cellent technical assistance, to Dr. Jean iVowakf o r automatic evaluation of protein patterns, and to Dr. Klaus Hinsch (Institute ofPharmacology, Berlin, FRG)for the iodination of marker proteins and mouse brain proteins. Received March 15, 1990
5 References
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