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Quantitation of protein and DNA in silver-stained agarose gels
ANALYTICAL
BIOCHEMISTRY
Quantitation
14&
178-l 82 ( 1984)
of Protein and DNA in Silver-Stained STEPHEN
Agarose
Gels
PEATS
Marine Colloids Div., FMC Corporation, BioProducts Department, 5 Maple Street, Rockland, Maine 04841 Received December 15, 1983 A silver stain for both proteins and DNA in agarose gels is described. Quantitation of proteins with this stain is possible, with individual proteins exhibiting characteristic responses, as observed with other stains. The advantage of the silver stain over Coomassie blue is its increased (50- to lOO-fold) sensitivity, which allows samples containing very low protein concentrations to be analyzed without prior concentration. This silver stain, when applied to DNA, is at least as sensitive as ethidium bromide, and gives a linear response for the type of DNA and fragment
sizesstudied. The most commonly used stain for the visualization and quantitation of proteins in agarose gels is Coomassie brilliant blue R-250. This stain is easy to use and gives reproducible results, but lacks sensitivity. The highly sensitive silver stains developed for polyacrylamide gels ( l-3) are not compatible with agarose due to excessive background staining. The first published silver stain used to detect electrophoretically separated proteins was that of Kerenyi and Gallyas (4). There have recently been a number of modifications of their stain for use with agarose gels (5-8), but little quantitative work has been published. Our purpose in carrying out the work described here was to demonstrate the general applicability of the silver stain to the quantitation of separated proteins in agarose gels, and to extend this to DNA gels, if possible. The finding of a means of eliminating artifactual spots was a prerequisite to carrying out quantitatiGe investigations, and has been reported elsewhere (9). Ethidium bromide, one of the most commonly used stains for nucleic acid detection, is a relatively simple stain to use. However, this stain necessitates the use of uv viewing equipment to visualize the stained bands, and associated photographic equipment if a permanent record is desired. Ethidium bromide
has the additional disadvantage of being a powerful mutagen. The silver stain presented here, when used to stain DNA, is at least as sensitive as ethidium bromide, and the dried gel on the plastic support film serves as a permanent record. MATERIALS
X 12 cm) were prepared from 1% agarose (SeaKem LE, FMC Corp.) in 0.05 M barbital buffer, pH 8.20, and cast on GelBond film (FMC Corp.) Protein standards (Sigma Chemical Co.) were dissolved in barbital buffer, and 2-~1 samples were applied directly to the gel through 10 X 1-mm slots in a plastic applicator mask. Electrophoresis, in the horizontal mode, was at a constant voltage of 180 V ( 16 V/cm) for 90 min. The gel was handled by using the extended border of the GelBond film to prevent surface contamination. Proteinjxative. All fixing and staining was in acid-cleaned (50% HNOj) glassware with constant agitation using a volume of 250 ml (gel volume:reagent volume = 1:22). Following electrophoresis, the gels were fixed for 30 min in a fixative which contained 500 ml methanol, 120 ml glacial acetic acid, and 50 g glycerol per liter. The gels were then dried for 90 min at 37°C in a forced-air oven. 178
0003-2697184 $3.00
AND METHODS
Protein electrophoresis.Gels (1 mm X 9 cm
QUANTITATION
OF
PROTEINS
AND
Coomassie stain. Oven-dried gels were washed with distilled water three times for 20 min each time, then placed overnight in the staining solution. The staining solution contained 0.50 g Coomassie brilliant blue R-250 (Bio-Rad Labs), 100 ml acetic acid, and 250 ml methanol per liter. Silver stain. Oven-dried gels were first washed in distilled water three times for 20 min each time, placed for 5 min in a solution that contained 20 g potassium ferrocyanide (&Fe [CN16. 3H20) per liter, and then given three 5-min washes in distilled water. To prepare the silver stain, equal volumes of Solutions A and B were mixed immediately prior to use. Solution B must be poured into a vigorously stirred Solution A. Solution A contains 50 g sodium carbonate (Na2C03) in 1 liter of distilled water, and is stable for about 2 to 3 weeks at room temperature. Solution B contains 2.00 g ammonium nitrate (NH,NOJ), 2.00 g silver nitrate (AgNO& 10.0 g tungstosilicic acid (SiO;!12W03 * 26H20), and 6.70 ml 37% formaldehyde (mixed in the order given) in 1 liter of distilled water, and is stable for about 1 week when stored in the dark at room temperature. The staining took approximately 10 min and was stopped by transferring the gel to a solution of 1% acetic acid for 5 min. The gel was then rinsed in distilled water and allowed to air dry. A black precipitate formed on the glass dish and, to some extent, on the back of the GelBond film. The deposit on the GelBond film can be removed following the acetic acid rinse by simply wiping with a paper towel. It should be cautioned that this deposit can create the impression of a dark background which may cause the operator to discontinue the staining process prematurely. DNA electrophoresis. Gels (1 mm X 7 cm X 16 cm) were prepared from 1% agarose (SeaKem LE, FMC Corp.) in 0.04 M Tris, 0.02 M sodium acetate, Cl.002 M Na2EDTA, pH 7.80, and cast on GelBond film. GelBond NF film was used when ethidium bromide was to be used as the stain. The electrophoresis
DNA
IN
SILVER-STAINED
GELS
179
was carried out in the vertical mode at 25 mA constant current for 3 h. Ethidium bromide stain. Gels were soaked in ethidium bromide (1 &ml) for 60 min, and then rinsed in distilled water for 5 min, viewed, and photographed (Polaroid Type 5 5, f/4.5, 5 min exposure) under uv light (302 nm), utilizing Wratten Nos. 2B and 29 gelatin filters. Silver stain. The gel was treated exactly as outlined for the silver stain of proteins with the following exceptions: (i) a gel volume to reagent volume ratio of 1:30 was used; (ii) No potassium ferrocyanide treatment, or the subsequent 3 X 5 min washes, were used; and (iii) 8.00 ml of 37% formaldehyde was used in Solution B. Densitometric scanning. Gels or photographic negatives were scanned at 600 nm using a Transidyne Model 2500. Peak areas were determined by both electronic integration and weighing. RESULTS
AND
DISCUSSIONS
Various fixatives have been used with the silver stain methodology. Willoughby and Lambert (8) used a trichloroacetic acid/sulfosalicylic acid/zinc sulfate fix for IgG detection. They concluded that pretreatment with potassium ferrocyanide was of no benefit, which is true with the fixative they used, as the addition of zinc sulfate may serve a similar purpose as the potassium ferrocyanide. With an acetic acid fixative, the potassium ferrocyanide is necessary. A trichloroacetic acid/ sulfosalicylic acid fixative has also been used when staining human palative salivary proteins ( 10) separated on agarose by isoelectric focusing. The concentration of the various components of the silver stain reagent can be varied (1 1), and will alter such parameters as the length of staining, grain size, and the formation of a precipitate in the staining reagent. The concentrations in this procedure result in staining times of approximately lo- 15 min,
180
STEPHEN
PEATS
FIG. 1. Agarose gels stained with (A) silver stain and (B) Coomassie blue. The proteins used are carbonic anhydrase, cat&se, and bovine serum albumin (closest to anode). The amounts of each of the three proteins by lane are 1, 1000 ng; 2, 500 ng; 3, 100 ng; 4, 50 ng; and 5, 10 ng.
small grain size, and a slow formation of precipitate, which avoids particles adhering to the gel surface and the need to use a fresh change of the silver stain reagent. The source of tungstosilicic acid can have an effect on both the characteristics and the cost of the stain (the tungstosilicic acid is the most expensive component of the reagent). Tungstosihcic acid from BDH (Poole, England) was preferred. The detection limits of the silver stain are of the order of 0.2 ng mm-‘, which is ap
FIG.
2. Peak area from de&tome.tric
proximately 100 times more sensitive than Coomassie blue (see Fig. 1). Protein concentrations in excess of 2.5 ng mm-’ result in OD greater than 4.0 (the upper limit of most commercial densitometers). The quantitative response of the silver stain (peak area vs protein loading) appears to be characteristic for each protein, Fig. 2. The linear range of the silver stain can be varied by altering the development time. Alternatively, when the protein concentrations cover a large range, the gels can be stained with Coomassie blue and
scan vs total amount of protein for six proteins.
QUANTITATION
A
12
OF PROTEINS
AND DNA IN SILVER-STAINED
3456
GELS
181
B 123456
FIG. 3. Agarose gel electrophoresis of bacteriophage X DNA Hi&II digest stained with (A) silver stain and (B) ethidium bromide. The amount of DNA loaded per lane is 1,400 ng; 2, 200 ng; 3, 100 ng; 4, 50 ng, 5, 25 ng, and 6, 12.5 ng. The fragment sizes (kbp) are 23.130; 9.419, 6.557; 4.371; 2.322; 2.028; and 0.564.
analyzed, and then those portions with no detectable protein can be subsequently silver stained ( 12). High protein concentrations give rise to a “halo effect,” common to all silver stains, which further complicates obtaining quantitative data. The ovalbumin band in Lanes 1 and 2 (Fig. 1A) demonstrates this effect, a lessdense center surrounded by a darker band. The cause of this effect is not known. Figure 3 is a comparison of a silver-stained pattern represented by the actual gel, and an ethidium bromide gel represented by a photographic negative obtained as outlined under Materials and Methods. The fact that a photographic negative was used for visual comparison and later for determination of quantitative response has to be considered. Effects such as reciprocity failure wih almost certaimy
,,,“a,
l t.tn
PE*K*RE* 320 280 240 ZOO too (20 80 40 20 40 60 80 MASSOFDNAFRABMEWT (ma)
tot
FIG. 4. Peak area from densitometric scan vs total amount of DNA in each bared. The plotted numbers refer to the lane number from which the values were obtained.
182
STEPHEN
contribute to the effectiveness of this comparison. Reduction in the physical size of the bands, resulting from the photographic procedures used for the ethidium bromide-stained gel, reduces the peak areas obtained. No attempt was made to normalize peak areas. A plot of peak area vs amount of DNA in each band is shown in Fig. 4. Accepting the limitations noted above, the silver stain appears to have potential for staining DNA quantitatively. These results are for the source of DNA noted and are in the molecular weight range 15 X lo6 to 0.37 X lo6 (23.1 to 0.56 kbp). A TuqI digest of 4X 174 RF DNA, molecular weight range 1.8 X lo6 to 0.06 X lo6 (2.82 to 0.06 kbp) electrophoretically separated on a 2% gel also stained satisfactorily. The silver stain results in a dried, stained gel which can serve as a permanent record. However, in producing a dry film, which is an integral part of the silver stain methodology, subsequent recovery of any of the DNA fractions is not possible. The main application of this stain would be for analytical, rather than preparative, separations. We have shown that the silver stain can be used to visualize both proteins and DNA in agarose gels in a quantitative manner. The stain appears to be very versatile, as it has also
PEATS
been shown (13) to stain proteins in polyacrylamide gels. No data, to our knowledge, have been published on using this stain for DNA separated on polyacrylamide gel, but this may also be possible. REFERENCES 1. Switzer, R. C., Merril, C. R., and Shifrin, S. (1979) Anal. Biochem. 98,231-237. 2. Oakley, B. R., Kirsch, D. R., and Morris, N. R. (1982) Anal. B&hem. 105, 361-363. 3. Merril, C. R., Goldman, D., and Van Keuren, M. L. (1982) Electrophoresis 3, 17-23. 4. Kerenyi, L., and Gallyas, F. (1972) Clin. Chim. Acta 38,465-467. 5. Verheecke, P. (1975) J. Neurol. 209, 59-63. 6. Karcher, D., Lowenthal, A., and Vantim, G. (1979) Acta Neurol. Belg. 19, 335-337. 7. Hiraoka, A., Isao, M., Muroa, O., Kawahara, N., Tomiraga, I., and Hattori, M. (1982) J. Neurosci. Methods 5, 22 l-226. 8. Willoughby, E. W., and Lambert, A. (1983) Anal. Biochem. 130,353-358. 9. Peats, S. (1983) BioTechniques 1, 154-156. 10. Shiba, A., Sano, K., Nakao, M., Kobayashi, K., and Igarashi, Y. (1983) Arch. Oral Biol. 28, 363-364. 11. GaIlyas, F. ( I97 1) Acta Morphologica Acad. Sci. Hung. 19,57-71. 12. Budowle, B. (1983) Presented at U. S. Electrophoresis ‘83, Boston, Mass. 13. Bartalos, L., Gallyas, F., and Merci, F. (1983) Ideggyogy sz. 36, 90-93.
BIOCHEMISTRY
Quantitation
14&
178-l 82 ( 1984)
of Protein and DNA in Silver-Stained STEPHEN
Agarose
Gels
PEATS
Marine Colloids Div., FMC Corporation, BioProducts Department, 5 Maple Street, Rockland, Maine 04841 Received December 15, 1983 A silver stain for both proteins and DNA in agarose gels is described. Quantitation of proteins with this stain is possible, with individual proteins exhibiting characteristic responses, as observed with other stains. The advantage of the silver stain over Coomassie blue is its increased (50- to lOO-fold) sensitivity, which allows samples containing very low protein concentrations to be analyzed without prior concentration. This silver stain, when applied to DNA, is at least as sensitive as ethidium bromide, and gives a linear response for the type of DNA and fragment
sizesstudied. The most commonly used stain for the visualization and quantitation of proteins in agarose gels is Coomassie brilliant blue R-250. This stain is easy to use and gives reproducible results, but lacks sensitivity. The highly sensitive silver stains developed for polyacrylamide gels ( l-3) are not compatible with agarose due to excessive background staining. The first published silver stain used to detect electrophoretically separated proteins was that of Kerenyi and Gallyas (4). There have recently been a number of modifications of their stain for use with agarose gels (5-8), but little quantitative work has been published. Our purpose in carrying out the work described here was to demonstrate the general applicability of the silver stain to the quantitation of separated proteins in agarose gels, and to extend this to DNA gels, if possible. The finding of a means of eliminating artifactual spots was a prerequisite to carrying out quantitatiGe investigations, and has been reported elsewhere (9). Ethidium bromide, one of the most commonly used stains for nucleic acid detection, is a relatively simple stain to use. However, this stain necessitates the use of uv viewing equipment to visualize the stained bands, and associated photographic equipment if a permanent record is desired. Ethidium bromide
has the additional disadvantage of being a powerful mutagen. The silver stain presented here, when used to stain DNA, is at least as sensitive as ethidium bromide, and the dried gel on the plastic support film serves as a permanent record. MATERIALS
X 12 cm) were prepared from 1% agarose (SeaKem LE, FMC Corp.) in 0.05 M barbital buffer, pH 8.20, and cast on GelBond film (FMC Corp.) Protein standards (Sigma Chemical Co.) were dissolved in barbital buffer, and 2-~1 samples were applied directly to the gel through 10 X 1-mm slots in a plastic applicator mask. Electrophoresis, in the horizontal mode, was at a constant voltage of 180 V ( 16 V/cm) for 90 min. The gel was handled by using the extended border of the GelBond film to prevent surface contamination. Proteinjxative. All fixing and staining was in acid-cleaned (50% HNOj) glassware with constant agitation using a volume of 250 ml (gel volume:reagent volume = 1:22). Following electrophoresis, the gels were fixed for 30 min in a fixative which contained 500 ml methanol, 120 ml glacial acetic acid, and 50 g glycerol per liter. The gels were then dried for 90 min at 37°C in a forced-air oven. 178
0003-2697184 $3.00
AND METHODS
Protein electrophoresis.Gels (1 mm X 9 cm
QUANTITATION
OF
PROTEINS
AND
Coomassie stain. Oven-dried gels were washed with distilled water three times for 20 min each time, then placed overnight in the staining solution. The staining solution contained 0.50 g Coomassie brilliant blue R-250 (Bio-Rad Labs), 100 ml acetic acid, and 250 ml methanol per liter. Silver stain. Oven-dried gels were first washed in distilled water three times for 20 min each time, placed for 5 min in a solution that contained 20 g potassium ferrocyanide (&Fe [CN16. 3H20) per liter, and then given three 5-min washes in distilled water. To prepare the silver stain, equal volumes of Solutions A and B were mixed immediately prior to use. Solution B must be poured into a vigorously stirred Solution A. Solution A contains 50 g sodium carbonate (Na2C03) in 1 liter of distilled water, and is stable for about 2 to 3 weeks at room temperature. Solution B contains 2.00 g ammonium nitrate (NH,NOJ), 2.00 g silver nitrate (AgNO& 10.0 g tungstosilicic acid (SiO;!12W03 * 26H20), and 6.70 ml 37% formaldehyde (mixed in the order given) in 1 liter of distilled water, and is stable for about 1 week when stored in the dark at room temperature. The staining took approximately 10 min and was stopped by transferring the gel to a solution of 1% acetic acid for 5 min. The gel was then rinsed in distilled water and allowed to air dry. A black precipitate formed on the glass dish and, to some extent, on the back of the GelBond film. The deposit on the GelBond film can be removed following the acetic acid rinse by simply wiping with a paper towel. It should be cautioned that this deposit can create the impression of a dark background which may cause the operator to discontinue the staining process prematurely. DNA electrophoresis. Gels (1 mm X 7 cm X 16 cm) were prepared from 1% agarose (SeaKem LE, FMC Corp.) in 0.04 M Tris, 0.02 M sodium acetate, Cl.002 M Na2EDTA, pH 7.80, and cast on GelBond film. GelBond NF film was used when ethidium bromide was to be used as the stain. The electrophoresis
DNA
IN
SILVER-STAINED
GELS
179
was carried out in the vertical mode at 25 mA constant current for 3 h. Ethidium bromide stain. Gels were soaked in ethidium bromide (1 &ml) for 60 min, and then rinsed in distilled water for 5 min, viewed, and photographed (Polaroid Type 5 5, f/4.5, 5 min exposure) under uv light (302 nm), utilizing Wratten Nos. 2B and 29 gelatin filters. Silver stain. The gel was treated exactly as outlined for the silver stain of proteins with the following exceptions: (i) a gel volume to reagent volume ratio of 1:30 was used; (ii) No potassium ferrocyanide treatment, or the subsequent 3 X 5 min washes, were used; and (iii) 8.00 ml of 37% formaldehyde was used in Solution B. Densitometric scanning. Gels or photographic negatives were scanned at 600 nm using a Transidyne Model 2500. Peak areas were determined by both electronic integration and weighing. RESULTS
AND
DISCUSSIONS
Various fixatives have been used with the silver stain methodology. Willoughby and Lambert (8) used a trichloroacetic acid/sulfosalicylic acid/zinc sulfate fix for IgG detection. They concluded that pretreatment with potassium ferrocyanide was of no benefit, which is true with the fixative they used, as the addition of zinc sulfate may serve a similar purpose as the potassium ferrocyanide. With an acetic acid fixative, the potassium ferrocyanide is necessary. A trichloroacetic acid/ sulfosalicylic acid fixative has also been used when staining human palative salivary proteins ( 10) separated on agarose by isoelectric focusing. The concentration of the various components of the silver stain reagent can be varied (1 1), and will alter such parameters as the length of staining, grain size, and the formation of a precipitate in the staining reagent. The concentrations in this procedure result in staining times of approximately lo- 15 min,
180
STEPHEN
PEATS
FIG. 1. Agarose gels stained with (A) silver stain and (B) Coomassie blue. The proteins used are carbonic anhydrase, cat&se, and bovine serum albumin (closest to anode). The amounts of each of the three proteins by lane are 1, 1000 ng; 2, 500 ng; 3, 100 ng; 4, 50 ng; and 5, 10 ng.
small grain size, and a slow formation of precipitate, which avoids particles adhering to the gel surface and the need to use a fresh change of the silver stain reagent. The source of tungstosilicic acid can have an effect on both the characteristics and the cost of the stain (the tungstosilicic acid is the most expensive component of the reagent). Tungstosihcic acid from BDH (Poole, England) was preferred. The detection limits of the silver stain are of the order of 0.2 ng mm-‘, which is ap
FIG.
2. Peak area from de&tome.tric
proximately 100 times more sensitive than Coomassie blue (see Fig. 1). Protein concentrations in excess of 2.5 ng mm-’ result in OD greater than 4.0 (the upper limit of most commercial densitometers). The quantitative response of the silver stain (peak area vs protein loading) appears to be characteristic for each protein, Fig. 2. The linear range of the silver stain can be varied by altering the development time. Alternatively, when the protein concentrations cover a large range, the gels can be stained with Coomassie blue and
scan vs total amount of protein for six proteins.
QUANTITATION
A
12
OF PROTEINS
AND DNA IN SILVER-STAINED
3456
GELS
181
B 123456
FIG. 3. Agarose gel electrophoresis of bacteriophage X DNA Hi&II digest stained with (A) silver stain and (B) ethidium bromide. The amount of DNA loaded per lane is 1,400 ng; 2, 200 ng; 3, 100 ng; 4, 50 ng, 5, 25 ng, and 6, 12.5 ng. The fragment sizes (kbp) are 23.130; 9.419, 6.557; 4.371; 2.322; 2.028; and 0.564.
analyzed, and then those portions with no detectable protein can be subsequently silver stained ( 12). High protein concentrations give rise to a “halo effect,” common to all silver stains, which further complicates obtaining quantitative data. The ovalbumin band in Lanes 1 and 2 (Fig. 1A) demonstrates this effect, a lessdense center surrounded by a darker band. The cause of this effect is not known. Figure 3 is a comparison of a silver-stained pattern represented by the actual gel, and an ethidium bromide gel represented by a photographic negative obtained as outlined under Materials and Methods. The fact that a photographic negative was used for visual comparison and later for determination of quantitative response has to be considered. Effects such as reciprocity failure wih almost certaimy
,,,“a,
l t.tn
PE*K*RE* 320 280 240 ZOO too (20 80 40 20 40 60 80 MASSOFDNAFRABMEWT (ma)
tot
FIG. 4. Peak area from densitometric scan vs total amount of DNA in each bared. The plotted numbers refer to the lane number from which the values were obtained.
182
STEPHEN
contribute to the effectiveness of this comparison. Reduction in the physical size of the bands, resulting from the photographic procedures used for the ethidium bromide-stained gel, reduces the peak areas obtained. No attempt was made to normalize peak areas. A plot of peak area vs amount of DNA in each band is shown in Fig. 4. Accepting the limitations noted above, the silver stain appears to have potential for staining DNA quantitatively. These results are for the source of DNA noted and are in the molecular weight range 15 X lo6 to 0.37 X lo6 (23.1 to 0.56 kbp). A TuqI digest of 4X 174 RF DNA, molecular weight range 1.8 X lo6 to 0.06 X lo6 (2.82 to 0.06 kbp) electrophoretically separated on a 2% gel also stained satisfactorily. The silver stain results in a dried, stained gel which can serve as a permanent record. However, in producing a dry film, which is an integral part of the silver stain methodology, subsequent recovery of any of the DNA fractions is not possible. The main application of this stain would be for analytical, rather than preparative, separations. We have shown that the silver stain can be used to visualize both proteins and DNA in agarose gels in a quantitative manner. The stain appears to be very versatile, as it has also
PEATS
been shown (13) to stain proteins in polyacrylamide gels. No data, to our knowledge, have been published on using this stain for DNA separated on polyacrylamide gel, but this may also be possible. REFERENCES 1. Switzer, R. C., Merril, C. R., and Shifrin, S. (1979) Anal. Biochem. 98,231-237. 2. Oakley, B. R., Kirsch, D. R., and Morris, N. R. (1982) Anal. B&hem. 105, 361-363. 3. Merril, C. R., Goldman, D., and Van Keuren, M. L. (1982) Electrophoresis 3, 17-23. 4. Kerenyi, L., and Gallyas, F. (1972) Clin. Chim. Acta 38,465-467. 5. Verheecke, P. (1975) J. Neurol. 209, 59-63. 6. Karcher, D., Lowenthal, A., and Vantim, G. (1979) Acta Neurol. Belg. 19, 335-337. 7. Hiraoka, A., Isao, M., Muroa, O., Kawahara, N., Tomiraga, I., and Hattori, M. (1982) J. Neurosci. Methods 5, 22 l-226. 8. Willoughby, E. W., and Lambert, A. (1983) Anal. Biochem. 130,353-358. 9. Peats, S. (1983) BioTechniques 1, 154-156. 10. Shiba, A., Sano, K., Nakao, M., Kobayashi, K., and Igarashi, Y. (1983) Arch. Oral Biol. 28, 363-364. 11. GaIlyas, F. ( I97 1) Acta Morphologica Acad. Sci. Hung. 19,57-71. 12. Budowle, B. (1983) Presented at U. S. Electrophoresis ‘83, Boston, Mass. 13. Bartalos, L., Gallyas, F., and Merci, F. (1983) Ideggyogy sz. 36, 90-93.