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Experimentally improved reliability of ultrasensitive silver staining of protein in polyacrylamide gels
ANALYTICAL
BIOCHEMISTRY
125,
96-99 (1982)
Experimentally Improved Reliability of Ultrasensitive Silver Staining of Protein in Polyacrylamide Gels MARGRET ESCHENBRUCHANDROBERT R. BARK' Friedrich Miescher-Institut,
Post Box 273, CH-4002 Basel, Switzerland
Received February 1, 1982 Variations of the ultrasensitive silver staining method of B. R. Oakley, D. R. Kirsch, and N. R. Morris (Anal. Biochem. 105, 361-363 (1980)) have been tested. It was established that the reliability of the method was greatly improved if (i) free silver was carefully washed out before reduction with formaldehyde; (ii) the extent of development was controlled by using methylamine to inactivate the formaldehyde; and (iii) the optimum quantity of ammonia, which was found to be 4 mol/mol of silver was used (this quantity was defined as that which titrates a particular amount of 1 N HCl). The time of preparation of the formaldehyde reducer was found not to be highly critical. In our hands the method can detect down to 0.1 ng of protein/S-mm slot.
of proteins to polyacrylamide by formaldehyde treatment (3), reliably allowed the detection of nanogram amounts of proteins against a clear background even in gels with polyacrylamide concentrations as high as 18%. This technique has been reviewed (4).
The ultrasensitive silver staining method developed by Switzer et al. (1) and simplified by Oakley et al. (2) permits detection of nanogram amounts of proteins in polyacrylamide gels and is therefore loo-fold more sensitive than the conventional staining method with Coomassie brilliant blue. A drawback of the method, however, is its unreliability. A number of laboratories experience the frequent but erratic concomitant or delayed development of a high, usually patchy, background that can mask the proteins. Apparently, some significant part of the procedure was not defined. We set out to identify the problem experimentally. The patches of background could be due to residual free ammoniacal silver that was not washed out adequately. Also, the delayed development could be prevented by directly inactivating the formaldehyde in the reducing agent rather than simply washing it out. Having tested and confirmed these possibilities, we were also able to test the requirements for “fresh” ammonia and formaldehyde. Our experiments lead to improvements that, in combination with chemical fixation
METHODS
Gels. For the staining experiment, 12.5% polyacrylamide-sodium dodecyl sulfate’ slab gels ( 1.6 mm thick) were prepared according to the method of Laemmli et al. (5). A group of six proteins (Bio-Rad protein standards) was used at concentrations of 1000, 100, 10, and 1 rig/protein/slot. The proteins were phosphorylase B (PB, M, 92,500); bovine serum albumin (BSA, M, 66,200); ovalbumin (OA, M, 45,000); carbonic anhydrase (CA, A4, 3 1,000); soybean trypsin inhibitor (STI, M, 21,500); and hen egg white lysozyme (EWL, M, 14,400). Staining. Formaldehyde fixation of proteins and simultaneous staining with Coom2 Abbreviations used: SDS, sodium dodecyl sulfate; PB, phosphorylase B; BSA, bovine serum albumin; OA, ovalbumin; CA, carbonic anhydrase; STI, soybean trypsin inhibitor; EWL, hen egg white lysozyme.
’ To whom all correspondence should be addressed. 0003-2697/82/130096-04$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
96
RELIABLE
SILVER STAINING
assie brilliant blue was performed as described by Steck et al. (3). After destaining, the gel was washed overnight in distilled water. It was then submitted to silver staining according to Oakley et al. (2) with the following alterations: (i) the glutaraldehyde fixation step was omitted because proteins had been fixed with formaldehyde; (ii) after treatment with ammoniacal silver solution the gel was washed 3 X 5 min in distilled water rather than 1 X 2 min as described by Oakley et al. (see Results); (iii) after development of protein bands the gel was quickly rinsed in Hz0 and placed immediately into a solution of 200 ml citric acid (0.05%) containing 100 ~1 aqueous methylamine (35%) (see Results); and (iv) before the gel was dried onto filter paper, the gel was soaked for 2 X 30 min in a solution containing 0.1 g citric acid, 146 ml water, 50 ml ethanol, 4 ml glycerol, and 100 ~1 aqueous methylamine (35%). EXPERIMENTAL
RESULTS
Removal of Residual Silver
A gel equilibrated in ammoniacal silver nitrate for 15 min was transferred to distilled water, and HCl was added to the spent wash water to give 0.05 M. There was a dense white precipitate of silver chloride. After the third wash there was no precipitate with HCl. It was concluded that the free silver had been washed out. Inactivation
of Formaldehyde
A gel containing various concentrations of proteins was cut into three equivalent parts immediately after development in formaldehyde citrate. The pieces were bathed in water alone, Oakley et al. (2) or either ethanolamine or methylamine in 0.05% citric acid. The piece of gel in the methylamine darkened slightly more, the piece in ethanolamine continued to darken for several minutes, and the piece in water continued to darken for over an hour. In further ex-
OF PROTEINS
97
periments gels dried without methylamine tended to darken during drying. Freshness of Formaldehyde Developer
A gel was sliced into four equal pieces and placed in 0.005% citric acid to which formaldehyde (to 0.05%) was added after the gel, or 2, 10, or 60 min before the gel. Development was stopped with methylamine at 8 min. All gels were stained to the same extent. Determination of the Optimum Ammonia Concentration in the Ammoniacal Silver Solution
The concentration of ammonia in an aqueous ammonia solution was determined by titration against standard 1.0 N HCl. Six solutions of ammoniacal silver reagent were then prepared using the aqueous ammonia at between 3 and 8 mol/mol of silver. Equivalent pieces of gel were placed in these solutions and washed and reduced as described under Methods. In the highest ammonia concentration no bands appeared even after 1 h of incubation. At the lowest concentration the bands appeared after 2 min, and when development was stopped after 8 min, most of the gel was brown. Other gels were also stopped after 8 min. The optimum sensitivity was obtained with 4 mol ammonia/ mol silver. This volume of aqueous ammonia titrates 18.4 ml of 1.0 N HCl to pH 4.7 and is suitable for use with 4 ml of silver nitrate solution (20 g AgNOj plus 100 ml water). Above this concentration of ammonia the bands stained more and more weakly, whereas below this concentration the background staining swamped the weaker protein bands. DISCUSSION
The use of a clearly defined ammonia concentration in the ammoniacal silver solution and the removal of surplus silver by differentiation in water reliably eliminated the occurrence of the erratic patchy background
98
ESCHENBRUCH
AND BiiRK
a
c
b
d
e
STI EWL
FIG. 1. Proteins silver stained using the improvements described in the text. (a) Stained only with Coomassie brilliant blue R-250; 1000 rig/protein. (b-e) Stained with Coomassie followed by silver; (b) 1000 ng, (c) 100 ng, (d) 10 ng, and (e) 1 rig/protein.
staining without decreasing the sensitivity of the method. As shown in Fig. 1, 1 ng of protein applied in a 5-mm slot can well be detected against a clear background. Overdevelopment of bands and background after reduction with formaldehyde has been eliminated by allowing surplus formaldehyde to react with a methylamine stopper solution; methylamine as a primary amine reacts with formaldehyde to form a Schiffs base. Oakley et al. (2) suggested the use of a photographic reducer for overstained gels, and Switzer et al. (1) describe a thiosulfate/cupric sulfate solution for the same purpose. In our hands, however, this latter solution produced a very rapid effect, and the reduction was difficult to control; after a clear background was obtained, destaining inside the gel continued despite rapid transfer into
water and resulted in a substantial decrease in sensitivity. Our method reliably produces a clear background and therefore makes the use of a photographic destainer unnecessary. A further advantage of this method is its usefulness for gels with polyacrylamide concentrations higher than 10%. Oakley et al. (2) observed an excessive background staining in gels with high polyacrylamide concentrations and suggested a pretreatment with a mixture of methanol/acetic acid before the fixation with glutaraldehyde. This, however, is not suitable for some basic proteins with low molecular weights as they are not precipitated by methanol/acetic acid and thus wash out. Formaldehyde fixation according to Steck et al. (3) followed by silver staining with the described modifications eliminates this problem.
RELIABLE
SILVER
STAINING
The ammonia concentration in the ammoniacal silver nitrate solution is critical. A 20% decrease of ammonia concentration results in rapid development with a noticeably higher background, whereas a 20% increase results in slow development with noticeably reduced sensitivity. To render the success of the method independent of the “freshness” referred to by Oakley et al. (2) and therefore the concentration of the ammonia solution available in the laboratory, the absolute amount of ammonia which is needed for optimum results (with 100 ml of reagent containing 4 ml silver nitrate solution (20 g AgN03 plus 100 ml water)) is the volume that titrates 18.4 ml of 1 N HCl to pH 4.7.
OF PROTEINS
99
At this optimum ammonia concentration some proteins give a clearly visible band in an SDS gel at 0.1 ng in a 5-mm slot. REFERENCES 1. Switzer, R. C. III, Merril, C. R., and Shifrin, S. (1979) Anal. Biochem. 98, 231-237. 2. Oakley, B. R., Kirsch, D. R., and Morris, N. R. (1980) Anal. Biochem. 105, 361-363. 3. Steck, G., Leuthard, P., and Btirk, R. R. (1980) Anal. Biochem. 107, 2 l-24. 4. Biirk, R. R., Eschenbruch, M., Steck, G., and Leuthard, P. (1983) in Methods in Enzymology: Enzyme Structure Part J (Hirs. C. H. W., and Timasheff, S. N., eds.), Academic Press, New York, in press. 5. Laemmli, U. K., (1970) Nature (London) 277,680685.
BIOCHEMISTRY
125,
96-99 (1982)
Experimentally Improved Reliability of Ultrasensitive Silver Staining of Protein in Polyacrylamide Gels MARGRET ESCHENBRUCHANDROBERT R. BARK' Friedrich Miescher-Institut,
Post Box 273, CH-4002 Basel, Switzerland
Received February 1, 1982 Variations of the ultrasensitive silver staining method of B. R. Oakley, D. R. Kirsch, and N. R. Morris (Anal. Biochem. 105, 361-363 (1980)) have been tested. It was established that the reliability of the method was greatly improved if (i) free silver was carefully washed out before reduction with formaldehyde; (ii) the extent of development was controlled by using methylamine to inactivate the formaldehyde; and (iii) the optimum quantity of ammonia, which was found to be 4 mol/mol of silver was used (this quantity was defined as that which titrates a particular amount of 1 N HCl). The time of preparation of the formaldehyde reducer was found not to be highly critical. In our hands the method can detect down to 0.1 ng of protein/S-mm slot.
of proteins to polyacrylamide by formaldehyde treatment (3), reliably allowed the detection of nanogram amounts of proteins against a clear background even in gels with polyacrylamide concentrations as high as 18%. This technique has been reviewed (4).
The ultrasensitive silver staining method developed by Switzer et al. (1) and simplified by Oakley et al. (2) permits detection of nanogram amounts of proteins in polyacrylamide gels and is therefore loo-fold more sensitive than the conventional staining method with Coomassie brilliant blue. A drawback of the method, however, is its unreliability. A number of laboratories experience the frequent but erratic concomitant or delayed development of a high, usually patchy, background that can mask the proteins. Apparently, some significant part of the procedure was not defined. We set out to identify the problem experimentally. The patches of background could be due to residual free ammoniacal silver that was not washed out adequately. Also, the delayed development could be prevented by directly inactivating the formaldehyde in the reducing agent rather than simply washing it out. Having tested and confirmed these possibilities, we were also able to test the requirements for “fresh” ammonia and formaldehyde. Our experiments lead to improvements that, in combination with chemical fixation
METHODS
Gels. For the staining experiment, 12.5% polyacrylamide-sodium dodecyl sulfate’ slab gels ( 1.6 mm thick) were prepared according to the method of Laemmli et al. (5). A group of six proteins (Bio-Rad protein standards) was used at concentrations of 1000, 100, 10, and 1 rig/protein/slot. The proteins were phosphorylase B (PB, M, 92,500); bovine serum albumin (BSA, M, 66,200); ovalbumin (OA, M, 45,000); carbonic anhydrase (CA, A4, 3 1,000); soybean trypsin inhibitor (STI, M, 21,500); and hen egg white lysozyme (EWL, M, 14,400). Staining. Formaldehyde fixation of proteins and simultaneous staining with Coom2 Abbreviations used: SDS, sodium dodecyl sulfate; PB, phosphorylase B; BSA, bovine serum albumin; OA, ovalbumin; CA, carbonic anhydrase; STI, soybean trypsin inhibitor; EWL, hen egg white lysozyme.
’ To whom all correspondence should be addressed. 0003-2697/82/130096-04$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
96
RELIABLE
SILVER STAINING
assie brilliant blue was performed as described by Steck et al. (3). After destaining, the gel was washed overnight in distilled water. It was then submitted to silver staining according to Oakley et al. (2) with the following alterations: (i) the glutaraldehyde fixation step was omitted because proteins had been fixed with formaldehyde; (ii) after treatment with ammoniacal silver solution the gel was washed 3 X 5 min in distilled water rather than 1 X 2 min as described by Oakley et al. (see Results); (iii) after development of protein bands the gel was quickly rinsed in Hz0 and placed immediately into a solution of 200 ml citric acid (0.05%) containing 100 ~1 aqueous methylamine (35%) (see Results); and (iv) before the gel was dried onto filter paper, the gel was soaked for 2 X 30 min in a solution containing 0.1 g citric acid, 146 ml water, 50 ml ethanol, 4 ml glycerol, and 100 ~1 aqueous methylamine (35%). EXPERIMENTAL
RESULTS
Removal of Residual Silver
A gel equilibrated in ammoniacal silver nitrate for 15 min was transferred to distilled water, and HCl was added to the spent wash water to give 0.05 M. There was a dense white precipitate of silver chloride. After the third wash there was no precipitate with HCl. It was concluded that the free silver had been washed out. Inactivation
of Formaldehyde
A gel containing various concentrations of proteins was cut into three equivalent parts immediately after development in formaldehyde citrate. The pieces were bathed in water alone, Oakley et al. (2) or either ethanolamine or methylamine in 0.05% citric acid. The piece of gel in the methylamine darkened slightly more, the piece in ethanolamine continued to darken for several minutes, and the piece in water continued to darken for over an hour. In further ex-
OF PROTEINS
97
periments gels dried without methylamine tended to darken during drying. Freshness of Formaldehyde Developer
A gel was sliced into four equal pieces and placed in 0.005% citric acid to which formaldehyde (to 0.05%) was added after the gel, or 2, 10, or 60 min before the gel. Development was stopped with methylamine at 8 min. All gels were stained to the same extent. Determination of the Optimum Ammonia Concentration in the Ammoniacal Silver Solution
The concentration of ammonia in an aqueous ammonia solution was determined by titration against standard 1.0 N HCl. Six solutions of ammoniacal silver reagent were then prepared using the aqueous ammonia at between 3 and 8 mol/mol of silver. Equivalent pieces of gel were placed in these solutions and washed and reduced as described under Methods. In the highest ammonia concentration no bands appeared even after 1 h of incubation. At the lowest concentration the bands appeared after 2 min, and when development was stopped after 8 min, most of the gel was brown. Other gels were also stopped after 8 min. The optimum sensitivity was obtained with 4 mol ammonia/ mol silver. This volume of aqueous ammonia titrates 18.4 ml of 1.0 N HCl to pH 4.7 and is suitable for use with 4 ml of silver nitrate solution (20 g AgNOj plus 100 ml water). Above this concentration of ammonia the bands stained more and more weakly, whereas below this concentration the background staining swamped the weaker protein bands. DISCUSSION
The use of a clearly defined ammonia concentration in the ammoniacal silver solution and the removal of surplus silver by differentiation in water reliably eliminated the occurrence of the erratic patchy background
98
ESCHENBRUCH
AND BiiRK
a
c
b
d
e
STI EWL
FIG. 1. Proteins silver stained using the improvements described in the text. (a) Stained only with Coomassie brilliant blue R-250; 1000 rig/protein. (b-e) Stained with Coomassie followed by silver; (b) 1000 ng, (c) 100 ng, (d) 10 ng, and (e) 1 rig/protein.
staining without decreasing the sensitivity of the method. As shown in Fig. 1, 1 ng of protein applied in a 5-mm slot can well be detected against a clear background. Overdevelopment of bands and background after reduction with formaldehyde has been eliminated by allowing surplus formaldehyde to react with a methylamine stopper solution; methylamine as a primary amine reacts with formaldehyde to form a Schiffs base. Oakley et al. (2) suggested the use of a photographic reducer for overstained gels, and Switzer et al. (1) describe a thiosulfate/cupric sulfate solution for the same purpose. In our hands, however, this latter solution produced a very rapid effect, and the reduction was difficult to control; after a clear background was obtained, destaining inside the gel continued despite rapid transfer into
water and resulted in a substantial decrease in sensitivity. Our method reliably produces a clear background and therefore makes the use of a photographic destainer unnecessary. A further advantage of this method is its usefulness for gels with polyacrylamide concentrations higher than 10%. Oakley et al. (2) observed an excessive background staining in gels with high polyacrylamide concentrations and suggested a pretreatment with a mixture of methanol/acetic acid before the fixation with glutaraldehyde. This, however, is not suitable for some basic proteins with low molecular weights as they are not precipitated by methanol/acetic acid and thus wash out. Formaldehyde fixation according to Steck et al. (3) followed by silver staining with the described modifications eliminates this problem.
RELIABLE
SILVER
STAINING
The ammonia concentration in the ammoniacal silver nitrate solution is critical. A 20% decrease of ammonia concentration results in rapid development with a noticeably higher background, whereas a 20% increase results in slow development with noticeably reduced sensitivity. To render the success of the method independent of the “freshness” referred to by Oakley et al. (2) and therefore the concentration of the ammonia solution available in the laboratory, the absolute amount of ammonia which is needed for optimum results (with 100 ml of reagent containing 4 ml silver nitrate solution (20 g AgN03 plus 100 ml water)) is the volume that titrates 18.4 ml of 1 N HCl to pH 4.7.
OF PROTEINS
99
At this optimum ammonia concentration some proteins give a clearly visible band in an SDS gel at 0.1 ng in a 5-mm slot. REFERENCES 1. Switzer, R. C. III, Merril, C. R., and Shifrin, S. (1979) Anal. Biochem. 98, 231-237. 2. Oakley, B. R., Kirsch, D. R., and Morris, N. R. (1980) Anal. Biochem. 105, 361-363. 3. Steck, G., Leuthard, P., and Btirk, R. R. (1980) Anal. Biochem. 107, 2 l-24. 4. Biirk, R. R., Eschenbruch, M., Steck, G., and Leuthard, P. (1983) in Methods in Enzymology: Enzyme Structure Part J (Hirs. C. H. W., and Timasheff, S. N., eds.), Academic Press, New York, in press. 5. Laemmli, U. K., (1970) Nature (London) 277,680685.