E1rcrrophowsi.P 1992, 13, 179-183
Jesper Christiansen Gunnar Houen Institute of Biochemical Genetics, University of Copenhagen
Compariron of different staining methods for PVDF membranes
179
Comparison of different staining methods for polyvinylidene difluoride membranes Several new staining methods for polyvinylidene difluoride membranes, including mercurochrome, silver and dimethylaminoazobenzene isothiocyanate staining were compared with Coomassie Brilliant Blue and gold staining. Of these, Coomassie was most versatile and completely compatible with ensuing microsequencing, immunostaining or other visualization methods, while gold and silver staining were more sensitive. Mercurochrome allows selective detection of sulfhydryl-containing proteins while dimethylaminoazobenzene isothiocyanate staining may allow quantitation of sequenceable protein.
Polyacrylamide gel electrophoreis (PAGE) and electroblotting of proteins from PAGE gels onto polymeric supports are important techniques for the identification, microsequencing and characterization of proteins [1-4]. This justifies comparison of existing staining methods [5,6], efforts to increase the sensitivity of staining methods [7-111, and the continued search for new staining methods [12-181 and new blotting membranes. In this report we describe several new methods for staining proteins on the recently introduced polyvinylidene difluoride (PVDE) membrane [ 19201 and compare these with existing methods. Silver nitrate, Amido Black, NaH,PO,, Na,HPO,, ammonium nitrate, Na,CO,, tungstosilicic acid, methanol, acetic acid, acetonitrile, pyridine, glutaraldehyde (25 O/o), formaldehyde (33 Yo), 33 O/o ammonia in water and Tween 20 were from Merck (Darmstadt, Germany). Mercurochrome and mercaptoethanol were from Fluka (Buchs, Switzerland). Sodium dodecyl sulfate (SDS), acrylamide, N,N-methylenebisacrylamide, N,N,NN-tetramethylethylenediamine and ammonium persulfate were from Bio-Rad (Richmond, USA). Bovine serum albumin (BSA), 3-amino-9-ethyl-carbazole and 3-[cyclohexylamino]-1-propanesulfonicacid (CAPS) were from Sigma (St. Louis, USA).Coomassie Brilliant Blue R-250 was from Serva (Heidelberg, Germany). Light Green was from BDH (Poole, England). 4-N,N-Dimethylaminoazobenzene-4'-isothiocyanate (Dabitc) was from Pierce (Oud Beijerland, The Netherlands). PVDF membranes were from Millipore (Bedford, USA). All water used was of Milli Q quality (Millipore). Trifluoroacetic acid (TFA) was from Applied Biosystems (Foster City, USA). Horseradish peroxidase-conjugated swine anti-rabbit antiserum was from DAKO (Copenhagen, Denmark). Rabbit anti-Dabitc antiserum was made by immunizing rabbits with BSA that had been derivatized with a large excess of Dabitc and dialyzed before immunization (SSI, Copenhagen, Denmark). SDS-PAGE was carried out as described by Laemmli [21] using 0.75 mm X 6 cm X 10 cm gels with 10% T, 2.6% C sep-
Correspondence: Dr. G . Houen, Institute of Biochemical Genetics, University of Copenhagen, (aster Farimagsgade 2A, DK-1353 Copenhagen, Denmark
arating gels and 4.6 O/o T,2.6% C stacking gels. Gels were cast and run using a Mighty Small Apparatus (Hoefer, San Francisco, USA). Preelectrophoresis of Laemmli gels for subsequent electroblotting onto PVDF membrane and microsequencing was done essentially as described by Houen et a/. [22], by subjecting gels to overnight preelectrophoresis in 375 m M Tris, pH 8.9,0.1% SDS, 20 mM mercaptopropionic acid at a current of 5 mA/gel. Samples were boiled in sample buffer with 40 mM dithiothreitol (DTT) and loaded directly on the gel or on top of a layer of 80% glycerol, 125 mM Tris, pH 6.7, 0.1% SDS. Electrophoresis was then performed with 20 mM mercaptopropionic acid in electrophoresis buffer. Electroblotting onto PVDF membrane was done with a semidry electroblotting apparatus (JKA Biotech, Copenhagen, Denmark) using 10 mM CAPS, pH 11,as transfer buffer at 1 mA/cm2for 1 h or 0.1 mA/cm2overnight. Membranes were wetted in methanol and then equilibrated in transfer buffer before electroblotting.The PVDFmembranes were washed 10 min in methanol and then 10 rnin in water prior to deposition of drops of protein solutions (5 pL) which were then allowed to dry. Membranes were then washed in 50% methanol before staining. Staining of polyacrylamide gels: Coomassie Brilliant Blue R-250: Gels were stained approximately 1 h in 40% methanol, 10% acetic acid with 2.5 mg Coomassie/mL, and then destained in 10 O/o acetic acid (2 X 15 min). Mercurochrome: Gels were immersed 30 rnin in 40% methanol, 10% acetic acid. Then they were stained for 30 rnin in 50% methanol with 5 mg mercurochrome/mL, and finally rinsed in 40 O/o methanol, 10% acetic acid. Silver staining: The protocol followed was essentially that described by De-Moreno et a/. [7]with minor modification: (i) Immerse gels for 10 rnin in 3.4 mM K,Cr,O,, 3.2 mM HNO,. (ii) Wash 3 X 10 min with deionized weater. (iii) Soak for 30 min in 12 mM silver nitrate. (iv) Wash 2 rnin with deionized water. (v) Develop three times (1,3, and 5 min) with 0.28 M sodium carbonate with 0.5 mL formaldehyde (33%) per liter. (vi) Stop development by immersing gels in 5 % acetic acid for 5 min. (vii) Rinse with deionized water. Combined Coomassie and silver staining: Gels were stained first with Coomassie as described above and then subjected to silver staining as described above.
Abbreviations: BSA, bovine serumalbumin; CAPS,3-[cyclohexylamino]- Staining of PVDF membranes: Membranes were treated 1-propanesulfonic acid; Dabitc, 4-N,N-dimethylaminoazobenzene-4'with 5 0 % methanol before staining. Coomassie Brilliant isothiocyanate; PAGE, polyacrylamide gel electrophoresis; PTH, phenyl thiohydantoin; PVDF, polyvinyl difluoride; SDS, sodium dodecyl sul- Blue R-250: Membranes were stained with a 0.1 O/o w/v solution of Coomassie Brilliant Blue R-250 in 50% methanol fate; TFA, trifluoroacetic acid
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for 10-30 min. Destaining was done in 40% methanol, 10°/o acetic acid, or in 50% methanol. For complete removal of dye, the membrane was washed with methanol. Mercurochrome: Membranes were immerscd in 50% methanol with 5 mg mercurochrome per mL for 1 h. After that they were briefly rinsed in 50% methanol. For some experiments the membranes were prewashed for 20 min in 1 mM mercaptoethanol in 50% methanol in order to reduce disulfide bonds and then washed for 20 min in water. This was later found not to be necessary for efficient staining, obviating destaining except for a brief rinse in 50% methanol or water. Amidoblack: Membranes were stained in 50% methanol with 5 mg .4midoblack per mLfor 10-30 min. Destaining was done i n 40% methanol, 10Vo acetic acid. Dabitc: Membranes were stained in a solution of pyridine: acetonitrile 1 :2 with 1 mg Dabitc per m L for 10-30 min and destained in acetonitrile. Dabitc + TFA: Membranes stained as stated above were air-dried and exposed to TFA over them. Light green: Membranes were stained in 50% methanol with 5 mg Light Green per mL for 10-30 min. Destaining was done in 40°/o methanol, 10% acetic acid. Silverstaining: Silver staining was done as described by De Moreno et al. [7] with the important modification that all steps were done in 50% acetonitrile instead of water. Silver intensification: Silver intensification of mercurochrome and silver-stained membranes was done by immersing membranes in Gallyas solution as described by Scopsi and Larsson [23]. Dabitc + rabbit anti-Dabitc: Membranes were stained with Dabitc as described above and then blocked in 1% BSA, 0.2% Tween 20 in 50 mM phosphate buffer, pH 7.2. They were then incubated 1 h with a rabbit antiserum against Dabitc, diluted 1:lOOO in l0/o BSA, 0.2% Tween 20 in phosphate buffer. After that they were washed 3 times for 10 min in 50 mM phosphate buffer and then incubated 1 h with peroxidaseconjugated swine anti-rabbit, diluted 1:lOOO in 1 Vo BSA, 0.2% Tween 20 in phosphate buffer. After washing 3 times in phosphate buffer, bound antibodies were visualized with 3-aniino-9-ethyl-carbazoleas described [23]. Gold staining: Membranes were immersed in a colloidal gold (15 nm) solution containing 120 mg HAuCI, per l L of 10 mM citrate buffer, pH 3 (the gold solution was a generous gift from Dr. V. Barkholt, The Danish Technical University). Amino acid analysis was done as described by Barkholt and Jensen [24] and Ploug et a / . [25]. Sequencing of protein on
membranes was done on an Applied Biosystem (Foster City, USA) Model 477 A sequencer with on-line analysis of phenylthiohydantoin (PTH-)amino acids using standard programs supplied with the sequencer. Figure 1 shows the results of comparing the sensitivity of various methods of staining proteins on PVDF membrane. Coomassie Brilliant Blue R-250 had a sensitivity down to 1-2 pmole BSA (125 ng). Mercurochrome. which produces red spots, also had a sensitivity down to 1-2 pmole BSA and could be used without destaining. This dye, which is also highly fluorescent, reacts preferentially with sulfhydryl-containing proteins (Fig. 2) and due to its content or Hg it is possible to enhance the sensitivity of this method further by silver enhancement. Amidoblack was slightly less sensitive while Light Green was even less sensitive. Dabitc staining produces yellow spots and had a sensitivity down to 20 pmole BSA.This sensitivity could be improved approximately tenfold by exposing the spots to TFA vapors, whereby more intense violet spots were produced. The sensitivity of this method could be even further improved to 0.5-1 pmole BSA using immunostaining with anti-Dabitc antibody. Silver staining with silver enhancement as described here had a sensitivity down to at least 0.5 pmole BSA. Direct application of the silver staining technique, as used with polyacrylamide gels, to a PVDF membrane is not possible due to the chemical structure of the membrane, which makes immobilized proteins inaccessible to the reagents. However, when all steps were performed in 50% acetonitrile. pink colored spots were produced that could be intensified further using Gallyas developer. Silver enhancement of mercurochrome-stained proteins also increased the sensitivity to 0.5 pmole BSA. Gold staining could also be carried out without destaining and had a sensitivity down to at least 0.5 pmole BSA. 0
Br
w
R - S H + H O - I igQ NaO
Br
__+
R-S-I
+H20
NaO
Br
F/,q//? 2. Keaction of mercurochrome with protein rulfydryl groups.
25 CLg ’ 0 pug 5 CLg
2500 ng 1250 ng 500 ng 250 ng 125 ng 50 ng 25ng
A
B
C
D
E
F
G
H
I
J
Ffgure I . Sensitivity of various staining methods. KSA in the indicated amounts were spotted on PVDF membranes and stained using the following procedures: A. Coomassie Brilliant Blue R-250; B, mercurochrome; C, Amidoblack; D, Dabitc: E, Dabitc + TFA; F, Light Green; G , silver staining +silver intensification; H, inercurochrome + silver intensification: 1. Dabitc +rabbit anti-Dabitc antiserum; J. goldstaining
Comparison of differcnt staining methods for I’VDF membranci
E l e c t m p h o w ~ i r1992. 13, 179-183
Figure 3 shows the results of applying some of the staining methods to human placental proteins separated by SDSPAGE and electrotransferred to PVDF membrane. Gold staining appears to be the most generally applicable stain since more proteins (especially of high molecular weight)
A
B
C
D
E
F
Figure 3. Staining of human placental proteins on PVDF membrane. A, Coomassie Brilliant Blue R-250; B, mercurochrome + silver intensification; C, gold staining; D, silver staining with silver intensification; E, Dabitc; F, Dabitc + TFA.
a
b
c
d
e
f
9
h
i
were visualized with it. For some proteins, silver staining with silver intensification was just as intense while mercurochrome with silver intensification was restricted to fewer proteins, a finding which can be ascribed to the selectivity of mercurochrome for sulfhydryl-containing proteins (Fig. 2). Coomassie Brilliant Blue R-250 was less sensitive than the metal-based stains, but was the most convenient method since the staining was fast and completely reversible, making subsequent removal of dye for microsequencing, iminunostaining or other staining methods possible. Thus, membranes could be stained with Coomassie and photographed, whereafter individual bands could be excised and destained for microsequencing, or whole lanes could be destained and subsequently immunostained or stained by other methods (Fig. 4). Dabitc staining was the least sensitive, but sequenceable amounts of proteins were clearly visible. Dabitc has the advantage of being an Edman reagent and therefore completely compatible with ensuing microsequencing, eliminating the risk of N-terminal blocking reactions. Serum albumin and P-lactoglobulin subjected to SDS-PAGE and electrotransfered to PVDF membrane were visualized by Dabitc staining and subjected to microsequencing (Table 1). One problem with Dabitc staining is that the reaction between Dabitc and amino groups is slow, making “after coupling” with phenylisothiocyanate (PITC) necessary in sequencing protocols [26]. This fact
i
k
I
Table 1. Results from microsequencing of bovine proteins after electrophoresis, electroblotting to PVDF membrane and staining by the indicated methods Staining method Protein Initial Residues Cycle, as identified yield identifiedlcycles Yield (pmole)
57
818 10110 313 414
1,Leu 105 103 35 199
2,Ile 88 104 29 507
3,Val 94 85 13 117
51
15/15
1,Asp 84
2,Thr 198
3,His 18
Coomassie Mercurochrome Gold Dabitc
PLG*)
89
-
88 30
-
Dabitc
BSA~)
181
a) From electrophoresis and electroblotting of bovine whey proteins (see Fig. 5) b ) From electrophoresis and electroblotting of commercial BSA (Sigma)
Figure 4. Staining of bovine whey pro,teins on PVDF membrane after electrophoresis, electroblotting, Coomassie staining, cutting out lanes, and destaining with methanol. Lanes a, g, j : molecular weight standard proteins stained with Coomassie. Lanes b, h , k : bovine whey proteins stained with Coomassie. Lanes c-f: immunostaining after blocking the membrane with skim milk powder (looh, lanes d, 0 or ovalbumin (loib, lanes c, e) in phosphate buffer. Following destaining with methanol, membranes were immersed in blocking solution for l h , and then incubated with antisera overnight at a dilution of 1:lOOO in blocking buffer. Membranes were then washed 3 times in phosphate buffer and then incubated l h with secondary antibody (peroxidase-conjugated swine anti-rabbit), diluted 1:1000 in blocking buffer. After a final washing in phosphate buffer (3 times) bound antibodies were visualized with 3-amino-9..ethyl carbazole as described [23]. Lane i: staining with mercurochrome after Coomassie staining. Lane 1: gold staining after Coomassie staining.
a
7
m
i
--2
i
‘ 7
a
E i e c r r o p h o r e ~ i !1992. 1.1.
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was reflected in the ready identification of PTH-Asp and PTH-Lys in the sequencing of BSA. However, the use of Dabitc for staining eliminates the risk of N-terminal blocking and the method would be ideal for quantitation of sequenceable protein [27] on blots and for subsequent application of Dabitc-based sequencing protocols. Of the more sensitive staining methods tested, Coomassie, mercurochrome and gold staining were compared with respect to microsequencing (Table 1). Sequencing of P-lactoglobulin after Coomassie Blue staining and removal of dye with methanol gave initial yields around 90% and n o artifacts were observed in the chromatograms (Fig. 5). Sequencing of P-lactoglobulin after mercurochrome staining without removal of the dye also gave initial yields around 90% and the only artifact observed was a peak eluting after Leu in the first cycles of the sequencing run. Sequencing of P-lactoglobulin after gold staining gave poor initial and repetitive yields. From the results presented here it is evident that Coomassie Brilliant Blue R-250 is the most versatile stain for PVDF membranes because it is completely reversible and highly sensitive. However, for some purposes, greater sensitivity may be required. in which case gold staining or silver staining/silver intensification may be used. These methods could also be used sequentially to obtain even higher sensitivity, as has been done in immunocytochemistry [23]. The failure of the silver staining method to stain directly on the PVDF membranes can be ascribed to the hydrophobic nature of the membrane, which makes the proteins inaccessible to the reagents. In 50% acetonitrile, however, the reactions, which rely on the generation of protein-bound aldehydes, which in turn reduce silver ions (Tollens reaction), can proceed at a sufficient rate. For selective detection of sulfhydryl-containing proteins, mercurochrome, alone or with silver intensification to improve sensitivity, can be used. For amino acid sequencing purposes, Dabitc staining has sufficient sensitivity and may allow a direct quantitation of the amount of sequenceable protein by performing the first cycle of Edman degradation manually on the membrane as described by Chang [26]. In conclusion, Coomassie Brilliant Blue has sufficient sensitivity to be used for most purposes with PVDF membranes, and can be removed completely to allow subsequent microsequencing or application of specialized staining methods, such as Dabitc staining, mercurochrome staining, immunostaining, silver staining, gold staining, etc.
Comparison of difrerenl staining methods for PVDF membranes
183
The authors wish to thank lnge Steiness,for help in preparing the manuscript and Pia Bach Kristensen .for technical assistance. Received February 6, 1991: in revised form November 18, 1991
References [l] Hames.B.D.and Rickwood,D.(Eds.), GelElectrophoresisofProteins IRL Press, Oxlbrd 1987. [2] Beisiegel, U.. Electrophoresis 1986, 7, 1-18. [3] Aebersold,R. H.,Teplow,D.B.,Hood,L. E.and Kent,S. B. H., J. Biol. Chein. 1986, 261, 4229-4238. 141 Simpson, R. J., Moritz, R. L., Begg, G . S . , Rubira, M. R. and Nice, E. C., Anal. Biochem. 1989, 177,221-236. [S] Wilson, C. M., Methods Enzyrnol. 1983, 91, 236-247. [6] Li,K. W., Geraerts. W. P. M.,van Elk, R. and Joosse, I.,Anal. Biochem. 1989, 182. 44-47. [7] De Morena, M. R., Smith, J. F. and Smith, K.V..Anal. Biodiern. 1985. 151. 466-470. [8] Li, K. W., Geraerts, W. P. M. and Joosse, J.,Anal. Biochein. 1988,174, 97-100. [9] Peisker, K., Electrophoresis 1988, Y, 236-238. [ 10) Neuhoff,V., Stamm, R. and Eibl, H., Electrophoresis 1985,6,427-448. [ I l l Domingo, A. and Marco, R., Anal. Biochem. 1989, 182, 176-181. [12] Selsted. M. E. and Becker, H. W., Anal. Biochern. 1986,155,270-274. [13] Lee, C., Levin. A . and Branton, D., Anal. Biochem. 1987, 166,3085IL.
[I41 Dzandu, J . K., Johnson, J. F. and Wise, G. E.. Anal. Biochrm. 1988. 174, 157-167. [15] Root, D. D. and Reisler, E., Anal. Biochem. 1989, 181, 250-253. [16] Samuel, D., Patt, R. J. and Abukenesha, R. A. J. Immunol. Methods 1988,107,217-224. [17] Hauber, R. and Geiger. R., J. Clin. Chem. Clin. Biochem. 1987, 25, 5 11-5 14. [18] Vera, J . C., Rivas, C. I., Cortes, P. A., Carcamo, J. 0.and Delgado, J., Anal. Blochem. 1988. 174, 38-45. [19] Pluskal,M. G.,Przekop, M. B.,Kavonian,M. R.,Vecoli,C. and Hicks, D. A , , BioTechniques 1986, 4, 272-283. [20] LeGendre, N . and Matsudaira, P., BioTechniques 1988, 6, 154-159. [21] Laemmli, U. K., Nurure 1970, 227, 680-685. [22] Houen, G . , Dando,T., Jensen,A. L. and Foltmann, B., J . Prot. Chrm. 1990, Y. 281-282. [23] Scopsi, L. and Larsson, L.-I., Histochernisfp 1986, 84, 221-230. [24] Barkholt, V. and Jensen, A. L., Anal. Biochem. 1989, 177, 318-322. [25] Ploug, M., Jensen, A. L. and Barkholl, V., Anal. Biochrm. 1989, 181, 33-39. [26] Wittmann-Liebold, B., Hirano, H. and Kimura, M., in: WittmannLiebold. B., Salnikov, I. and Erdmann, V. A. (Eds.), Advanced Methads in Protein Microsequence Anulysi.r, Springer-Verlag, Berlin 1986 PP. 77-90. 1271 Chane. " J.-Y.. Anal. Biochem. 1988. 170. 542-556. L
1
Jesper Christiansen Gunnar Houen Institute of Biochemical Genetics, University of Copenhagen
Compariron of different staining methods for PVDF membranes
179
Comparison of different staining methods for polyvinylidene difluoride membranes Several new staining methods for polyvinylidene difluoride membranes, including mercurochrome, silver and dimethylaminoazobenzene isothiocyanate staining were compared with Coomassie Brilliant Blue and gold staining. Of these, Coomassie was most versatile and completely compatible with ensuing microsequencing, immunostaining or other visualization methods, while gold and silver staining were more sensitive. Mercurochrome allows selective detection of sulfhydryl-containing proteins while dimethylaminoazobenzene isothiocyanate staining may allow quantitation of sequenceable protein.
Polyacrylamide gel electrophoreis (PAGE) and electroblotting of proteins from PAGE gels onto polymeric supports are important techniques for the identification, microsequencing and characterization of proteins [1-4]. This justifies comparison of existing staining methods [5,6], efforts to increase the sensitivity of staining methods [7-111, and the continued search for new staining methods [12-181 and new blotting membranes. In this report we describe several new methods for staining proteins on the recently introduced polyvinylidene difluoride (PVDE) membrane [ 19201 and compare these with existing methods. Silver nitrate, Amido Black, NaH,PO,, Na,HPO,, ammonium nitrate, Na,CO,, tungstosilicic acid, methanol, acetic acid, acetonitrile, pyridine, glutaraldehyde (25 O/o), formaldehyde (33 Yo), 33 O/o ammonia in water and Tween 20 were from Merck (Darmstadt, Germany). Mercurochrome and mercaptoethanol were from Fluka (Buchs, Switzerland). Sodium dodecyl sulfate (SDS), acrylamide, N,N-methylenebisacrylamide, N,N,NN-tetramethylethylenediamine and ammonium persulfate were from Bio-Rad (Richmond, USA). Bovine serum albumin (BSA), 3-amino-9-ethyl-carbazole and 3-[cyclohexylamino]-1-propanesulfonicacid (CAPS) were from Sigma (St. Louis, USA).Coomassie Brilliant Blue R-250 was from Serva (Heidelberg, Germany). Light Green was from BDH (Poole, England). 4-N,N-Dimethylaminoazobenzene-4'-isothiocyanate (Dabitc) was from Pierce (Oud Beijerland, The Netherlands). PVDF membranes were from Millipore (Bedford, USA). All water used was of Milli Q quality (Millipore). Trifluoroacetic acid (TFA) was from Applied Biosystems (Foster City, USA). Horseradish peroxidase-conjugated swine anti-rabbit antiserum was from DAKO (Copenhagen, Denmark). Rabbit anti-Dabitc antiserum was made by immunizing rabbits with BSA that had been derivatized with a large excess of Dabitc and dialyzed before immunization (SSI, Copenhagen, Denmark). SDS-PAGE was carried out as described by Laemmli [21] using 0.75 mm X 6 cm X 10 cm gels with 10% T, 2.6% C sep-
Correspondence: Dr. G . Houen, Institute of Biochemical Genetics, University of Copenhagen, (aster Farimagsgade 2A, DK-1353 Copenhagen, Denmark
arating gels and 4.6 O/o T,2.6% C stacking gels. Gels were cast and run using a Mighty Small Apparatus (Hoefer, San Francisco, USA). Preelectrophoresis of Laemmli gels for subsequent electroblotting onto PVDF membrane and microsequencing was done essentially as described by Houen et a/. [22], by subjecting gels to overnight preelectrophoresis in 375 m M Tris, pH 8.9,0.1% SDS, 20 mM mercaptopropionic acid at a current of 5 mA/gel. Samples were boiled in sample buffer with 40 mM dithiothreitol (DTT) and loaded directly on the gel or on top of a layer of 80% glycerol, 125 mM Tris, pH 6.7, 0.1% SDS. Electrophoresis was then performed with 20 mM mercaptopropionic acid in electrophoresis buffer. Electroblotting onto PVDF membrane was done with a semidry electroblotting apparatus (JKA Biotech, Copenhagen, Denmark) using 10 mM CAPS, pH 11,as transfer buffer at 1 mA/cm2for 1 h or 0.1 mA/cm2overnight. Membranes were wetted in methanol and then equilibrated in transfer buffer before electroblotting.The PVDFmembranes were washed 10 min in methanol and then 10 rnin in water prior to deposition of drops of protein solutions (5 pL) which were then allowed to dry. Membranes were then washed in 50% methanol before staining. Staining of polyacrylamide gels: Coomassie Brilliant Blue R-250: Gels were stained approximately 1 h in 40% methanol, 10% acetic acid with 2.5 mg Coomassie/mL, and then destained in 10 O/o acetic acid (2 X 15 min). Mercurochrome: Gels were immersed 30 rnin in 40% methanol, 10% acetic acid. Then they were stained for 30 rnin in 50% methanol with 5 mg mercurochrome/mL, and finally rinsed in 40 O/o methanol, 10% acetic acid. Silver staining: The protocol followed was essentially that described by De-Moreno et a/. [7]with minor modification: (i) Immerse gels for 10 rnin in 3.4 mM K,Cr,O,, 3.2 mM HNO,. (ii) Wash 3 X 10 min with deionized weater. (iii) Soak for 30 min in 12 mM silver nitrate. (iv) Wash 2 rnin with deionized water. (v) Develop three times (1,3, and 5 min) with 0.28 M sodium carbonate with 0.5 mL formaldehyde (33%) per liter. (vi) Stop development by immersing gels in 5 % acetic acid for 5 min. (vii) Rinse with deionized water. Combined Coomassie and silver staining: Gels were stained first with Coomassie as described above and then subjected to silver staining as described above.
Abbreviations: BSA, bovine serumalbumin; CAPS,3-[cyclohexylamino]- Staining of PVDF membranes: Membranes were treated 1-propanesulfonic acid; Dabitc, 4-N,N-dimethylaminoazobenzene-4'with 5 0 % methanol before staining. Coomassie Brilliant isothiocyanate; PAGE, polyacrylamide gel electrophoresis; PTH, phenyl thiohydantoin; PVDF, polyvinyl difluoride; SDS, sodium dodecyl sul- Blue R-250: Membranes were stained with a 0.1 O/o w/v solution of Coomassie Brilliant Blue R-250 in 50% methanol fate; TFA, trifluoroacetic acid
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.1. (‘linsllanscn and G . Ilouen
for 10-30 min. Destaining was done in 40% methanol, 10°/o acetic acid, or in 50% methanol. For complete removal of dye, the membrane was washed with methanol. Mercurochrome: Membranes were immerscd in 50% methanol with 5 mg mercurochrome per mL for 1 h. After that they were briefly rinsed in 50% methanol. For some experiments the membranes were prewashed for 20 min in 1 mM mercaptoethanol in 50% methanol in order to reduce disulfide bonds and then washed for 20 min in water. This was later found not to be necessary for efficient staining, obviating destaining except for a brief rinse in 50% methanol or water. Amidoblack: Membranes were stained in 50% methanol with 5 mg .4midoblack per mLfor 10-30 min. Destaining was done i n 40% methanol, 10Vo acetic acid. Dabitc: Membranes were stained in a solution of pyridine: acetonitrile 1 :2 with 1 mg Dabitc per m L for 10-30 min and destained in acetonitrile. Dabitc + TFA: Membranes stained as stated above were air-dried and exposed to TFA over them. Light green: Membranes were stained in 50% methanol with 5 mg Light Green per mL for 10-30 min. Destaining was done in 40°/o methanol, 10% acetic acid. Silverstaining: Silver staining was done as described by De Moreno et al. [7] with the important modification that all steps were done in 50% acetonitrile instead of water. Silver intensification: Silver intensification of mercurochrome and silver-stained membranes was done by immersing membranes in Gallyas solution as described by Scopsi and Larsson [23]. Dabitc + rabbit anti-Dabitc: Membranes were stained with Dabitc as described above and then blocked in 1% BSA, 0.2% Tween 20 in 50 mM phosphate buffer, pH 7.2. They were then incubated 1 h with a rabbit antiserum against Dabitc, diluted 1:lOOO in l0/o BSA, 0.2% Tween 20 in phosphate buffer. After that they were washed 3 times for 10 min in 50 mM phosphate buffer and then incubated 1 h with peroxidaseconjugated swine anti-rabbit, diluted 1:lOOO in 1 Vo BSA, 0.2% Tween 20 in phosphate buffer. After washing 3 times in phosphate buffer, bound antibodies were visualized with 3-aniino-9-ethyl-carbazoleas described [23]. Gold staining: Membranes were immersed in a colloidal gold (15 nm) solution containing 120 mg HAuCI, per l L of 10 mM citrate buffer, pH 3 (the gold solution was a generous gift from Dr. V. Barkholt, The Danish Technical University). Amino acid analysis was done as described by Barkholt and Jensen [24] and Ploug et a / . [25]. Sequencing of protein on
membranes was done on an Applied Biosystem (Foster City, USA) Model 477 A sequencer with on-line analysis of phenylthiohydantoin (PTH-)amino acids using standard programs supplied with the sequencer. Figure 1 shows the results of comparing the sensitivity of various methods of staining proteins on PVDF membrane. Coomassie Brilliant Blue R-250 had a sensitivity down to 1-2 pmole BSA (125 ng). Mercurochrome. which produces red spots, also had a sensitivity down to 1-2 pmole BSA and could be used without destaining. This dye, which is also highly fluorescent, reacts preferentially with sulfhydryl-containing proteins (Fig. 2) and due to its content or Hg it is possible to enhance the sensitivity of this method further by silver enhancement. Amidoblack was slightly less sensitive while Light Green was even less sensitive. Dabitc staining produces yellow spots and had a sensitivity down to 20 pmole BSA.This sensitivity could be improved approximately tenfold by exposing the spots to TFA vapors, whereby more intense violet spots were produced. The sensitivity of this method could be even further improved to 0.5-1 pmole BSA using immunostaining with anti-Dabitc antibody. Silver staining with silver enhancement as described here had a sensitivity down to at least 0.5 pmole BSA. Direct application of the silver staining technique, as used with polyacrylamide gels, to a PVDF membrane is not possible due to the chemical structure of the membrane, which makes immobilized proteins inaccessible to the reagents. However, when all steps were performed in 50% acetonitrile. pink colored spots were produced that could be intensified further using Gallyas developer. Silver enhancement of mercurochrome-stained proteins also increased the sensitivity to 0.5 pmole BSA. Gold staining could also be carried out without destaining and had a sensitivity down to at least 0.5 pmole BSA. 0
Br
w
R - S H + H O - I igQ NaO
Br
__+
R-S-I
+H20
NaO
Br
F/,q//? 2. Keaction of mercurochrome with protein rulfydryl groups.
25 CLg ’ 0 pug 5 CLg
2500 ng 1250 ng 500 ng 250 ng 125 ng 50 ng 25ng
A
B
C
D
E
F
G
H
I
J
Ffgure I . Sensitivity of various staining methods. KSA in the indicated amounts were spotted on PVDF membranes and stained using the following procedures: A. Coomassie Brilliant Blue R-250; B, mercurochrome; C, Amidoblack; D, Dabitc: E, Dabitc + TFA; F, Light Green; G , silver staining +silver intensification; H, inercurochrome + silver intensification: 1. Dabitc +rabbit anti-Dabitc antiserum; J. goldstaining
Comparison of differcnt staining methods for I’VDF membranci
E l e c t m p h o w ~ i r1992. 13, 179-183
Figure 3 shows the results of applying some of the staining methods to human placental proteins separated by SDSPAGE and electrotransferred to PVDF membrane. Gold staining appears to be the most generally applicable stain since more proteins (especially of high molecular weight)
A
B
C
D
E
F
Figure 3. Staining of human placental proteins on PVDF membrane. A, Coomassie Brilliant Blue R-250; B, mercurochrome + silver intensification; C, gold staining; D, silver staining with silver intensification; E, Dabitc; F, Dabitc + TFA.
a
b
c
d
e
f
9
h
i
were visualized with it. For some proteins, silver staining with silver intensification was just as intense while mercurochrome with silver intensification was restricted to fewer proteins, a finding which can be ascribed to the selectivity of mercurochrome for sulfhydryl-containing proteins (Fig. 2). Coomassie Brilliant Blue R-250 was less sensitive than the metal-based stains, but was the most convenient method since the staining was fast and completely reversible, making subsequent removal of dye for microsequencing, iminunostaining or other staining methods possible. Thus, membranes could be stained with Coomassie and photographed, whereafter individual bands could be excised and destained for microsequencing, or whole lanes could be destained and subsequently immunostained or stained by other methods (Fig. 4). Dabitc staining was the least sensitive, but sequenceable amounts of proteins were clearly visible. Dabitc has the advantage of being an Edman reagent and therefore completely compatible with ensuing microsequencing, eliminating the risk of N-terminal blocking reactions. Serum albumin and P-lactoglobulin subjected to SDS-PAGE and electrotransfered to PVDF membrane were visualized by Dabitc staining and subjected to microsequencing (Table 1). One problem with Dabitc staining is that the reaction between Dabitc and amino groups is slow, making “after coupling” with phenylisothiocyanate (PITC) necessary in sequencing protocols [26]. This fact
i
k
I
Table 1. Results from microsequencing of bovine proteins after electrophoresis, electroblotting to PVDF membrane and staining by the indicated methods Staining method Protein Initial Residues Cycle, as identified yield identifiedlcycles Yield (pmole)
57
818 10110 313 414
1,Leu 105 103 35 199
2,Ile 88 104 29 507
3,Val 94 85 13 117
51
15/15
1,Asp 84
2,Thr 198
3,His 18
Coomassie Mercurochrome Gold Dabitc
PLG*)
89
-
88 30
-
Dabitc
BSA~)
181
a) From electrophoresis and electroblotting of bovine whey proteins (see Fig. 5) b ) From electrophoresis and electroblotting of commercial BSA (Sigma)
Figure 4. Staining of bovine whey pro,teins on PVDF membrane after electrophoresis, electroblotting, Coomassie staining, cutting out lanes, and destaining with methanol. Lanes a, g, j : molecular weight standard proteins stained with Coomassie. Lanes b, h , k : bovine whey proteins stained with Coomassie. Lanes c-f: immunostaining after blocking the membrane with skim milk powder (looh, lanes d, 0 or ovalbumin (loib, lanes c, e) in phosphate buffer. Following destaining with methanol, membranes were immersed in blocking solution for l h , and then incubated with antisera overnight at a dilution of 1:lOOO in blocking buffer. Membranes were then washed 3 times in phosphate buffer and then incubated l h with secondary antibody (peroxidase-conjugated swine anti-rabbit), diluted 1:1000 in blocking buffer. After a final washing in phosphate buffer (3 times) bound antibodies were visualized with 3-amino-9..ethyl carbazole as described [23]. Lane i: staining with mercurochrome after Coomassie staining. Lane 1: gold staining after Coomassie staining.
a
7
m
i
--2
i
‘ 7
a
E i e c r r o p h o r e ~ i !1992. 1.1.
119-1 83
was reflected in the ready identification of PTH-Asp and PTH-Lys in the sequencing of BSA. However, the use of Dabitc for staining eliminates the risk of N-terminal blocking and the method would be ideal for quantitation of sequenceable protein [27] on blots and for subsequent application of Dabitc-based sequencing protocols. Of the more sensitive staining methods tested, Coomassie, mercurochrome and gold staining were compared with respect to microsequencing (Table 1). Sequencing of P-lactoglobulin after Coomassie Blue staining and removal of dye with methanol gave initial yields around 90% and n o artifacts were observed in the chromatograms (Fig. 5). Sequencing of P-lactoglobulin after mercurochrome staining without removal of the dye also gave initial yields around 90% and the only artifact observed was a peak eluting after Leu in the first cycles of the sequencing run. Sequencing of P-lactoglobulin after gold staining gave poor initial and repetitive yields. From the results presented here it is evident that Coomassie Brilliant Blue R-250 is the most versatile stain for PVDF membranes because it is completely reversible and highly sensitive. However, for some purposes, greater sensitivity may be required. in which case gold staining or silver staining/silver intensification may be used. These methods could also be used sequentially to obtain even higher sensitivity, as has been done in immunocytochemistry [23]. The failure of the silver staining method to stain directly on the PVDF membranes can be ascribed to the hydrophobic nature of the membrane, which makes the proteins inaccessible to the reagents. In 50% acetonitrile, however, the reactions, which rely on the generation of protein-bound aldehydes, which in turn reduce silver ions (Tollens reaction), can proceed at a sufficient rate. For selective detection of sulfhydryl-containing proteins, mercurochrome, alone or with silver intensification to improve sensitivity, can be used. For amino acid sequencing purposes, Dabitc staining has sufficient sensitivity and may allow a direct quantitation of the amount of sequenceable protein by performing the first cycle of Edman degradation manually on the membrane as described by Chang [26]. In conclusion, Coomassie Brilliant Blue has sufficient sensitivity to be used for most purposes with PVDF membranes, and can be removed completely to allow subsequent microsequencing or application of specialized staining methods, such as Dabitc staining, mercurochrome staining, immunostaining, silver staining, gold staining, etc.
Comparison of difrerenl staining methods for PVDF membranes
183
The authors wish to thank lnge Steiness,for help in preparing the manuscript and Pia Bach Kristensen .for technical assistance. Received February 6, 1991: in revised form November 18, 1991
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[I41 Dzandu, J . K., Johnson, J. F. and Wise, G. E.. Anal. Biochrm. 1988. 174, 157-167. [15] Root, D. D. and Reisler, E., Anal. Biochem. 1989, 181, 250-253. [16] Samuel, D., Patt, R. J. and Abukenesha, R. A. J. Immunol. Methods 1988,107,217-224. [17] Hauber, R. and Geiger. R., J. Clin. Chem. Clin. Biochem. 1987, 25, 5 11-5 14. [18] Vera, J . C., Rivas, C. I., Cortes, P. A., Carcamo, J. 0.and Delgado, J., Anal. Blochem. 1988. 174, 38-45. [19] Pluskal,M. G.,Przekop, M. B.,Kavonian,M. R.,Vecoli,C. and Hicks, D. A , , BioTechniques 1986, 4, 272-283. [20] LeGendre, N . and Matsudaira, P., BioTechniques 1988, 6, 154-159. [21] Laemmli, U. K., Nurure 1970, 227, 680-685. [22] Houen, G . , Dando,T., Jensen,A. L. and Foltmann, B., J . Prot. Chrm. 1990, Y. 281-282. [23] Scopsi, L. and Larsson, L.-I., Histochernisfp 1986, 84, 221-230. [24] Barkholt, V. and Jensen, A. L., Anal. Biochem. 1989, 177, 318-322. [25] Ploug, M., Jensen, A. L. and Barkholl, V., Anal. Biochrm. 1989, 181, 33-39. [26] Wittmann-Liebold, B., Hirano, H. and Kimura, M., in: WittmannLiebold. B., Salnikov, I. and Erdmann, V. A. (Eds.), Advanced Methads in Protein Microsequence Anulysi.r, Springer-Verlag, Berlin 1986 PP. 77-90. 1271 Chane. " J.-Y.. Anal. Biochem. 1988. 170. 542-556. L
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