ANALYTICAL
BIOCHEMISTRY
72, 366-371
(1976)
Recovery of SDS-Proteins from Polyacrylamide Gels by Electrophoresis into Hydroxylapatite BARRY R. ZIOLAANDDOUGLAS Department
of Biochemistry,
University of Alberta,
G. SCRABA Edmonton, Alberta
Received May 16, 1975; accepted November
T6G 2H7
19, 1975
A method is described which permits the purification of proteins in quantities necessary for physicochemical characterization. The protein to be purified is separated from contaminants by electrophoresis in a number of sodium dodecyl sulfate (SDS)-containing polyacrylamide disc gels. The region of each gel which contains the desired protein band is then cut out and the slices are stacked one above another in a tube. After the protein is electrophoresed from the gel slices into a bed of hydroxylapatite, it is eluted from the hydroxylapatite with 0.5 M sodium phosphate (pH 6.4) containing 0.1% SDS and 1 mM dithiothreitol. Protein recovery is greater than 90%.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) has gained wide acceptance as an analytical tool for assaying protein at progressive stages of purification. The application of SDS-PAGE to preparative procedures, however, has been hampered by dilution of the protein upon elution or extraction from gels and by contamination of the recovered protein by a gel impurity, which is believed to be linear polyacrylate (1). During the course of our studies on the Mengo encephalomyelitis virus capsid proteins (2), we occasionally encountered difficulty in separating two of the four virus structural polypeptides (i.e., the CYand y chains, of molecular weights 32,500 and 23,700, respectively) using the SDShydroxylapatite chromatography method of Moss and Rosenblum (3). As a result, we have developed a method for the quantitative recovery of these two proteins following their separation by SDS-PAGE. The method overcomes the protein dilution problem of most preparative electrophoresis systems; and in addition, uv absorption spectra of the recovered protein indicate that there is very little contamination by gel impurities. This preparative procedure should prove applicable to any polypeptide isolation where SDS-PAGE can be used for separation. For the purpose of illustration, a description is given of the isolation of a-chymotrypsinogen A (cu-chtgn A) from a mixture of proteins. METHODS Materials, Lactic dehydrogenase (LDH) and a-chtgn A were obtained from Sigma Chemical Co., carboxypeptidase A (CBP A) and a-chymo366 Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
PROTEIN
ISOLATION
METHOD
367
trypsin (a-cht) were from Worthington Biochemicals, and myoglobin (Myo) was from Schwarz/Mann Biochemicals.’ The dye Remazol Brilliant Blue R (RBBR) was purchased from Calbiochem. Acrylamide and N,N’methylenebisacrylamide were obtained from Eastman Organic Chemicals and recrystallized according to the procedure described by Loening (4). Dithiothreitol (DTT), technical grade SDS, and hydroxylapatite (HA) in powder form (Bio-Gel HTP) were purchased from Sigma Chemical Co., Matheson. Coleman, and Bell, and Bio-Rad Laboratories, respectively. All other chemicals were reagent grade. SDS-Polyacrylamide gel electrophoresis. All gels consisted of 10% acryalamide, 0.2% N,N’-methylenebisacrylamide, 0.1% SDS, and 0.1 M sodium phosphate (pH 7.2). Polymerization was catalyzed by N,N,N’N’tetramethylethylenediamine and ammonium persulfate, both at final concentrations of 0.075%. The gels were pre-electrophoresed for a minimum of 1 hr. LDH, cY-chtgn A, and a-cht were each dissolved in 0.01 M sodium phosphate (pH 7.2) containing 2% SDS and 10% glycerol. CBP A and Myo were stained by the protein-RBBR coupling procedure of Griffith (5) and similarly dissolved. A mixture containing 1.5 mg each of LDH and cY-chtgn A, and 3.0 mg of a-cht was made 5% with respect to P-mercaptoethanol (final volume, 770 ~1) and heated at 100°C for 5 min (under these conditions, a-cht dissociates to its constituent A, B, and C chains. Aliquots (150 ~1) were layered onto each of five SDS-polyacrylamide preparative gels (1 x 6 cm) directly beneath the electrophoresis buffer (0.1 M sodium phosphate, pH 7.2, containing 0.1% SDS). A 150 ~1 sample containing 300 pg of each of the prestained marker proteins, CPB A-RBBR and Myo-RBBR, was similarly prepared and layered onto a sixth gel. Electrophoresis was at 9-10 mA/gel for 10 hr. Following electrophoresis, the marker gel was aligned with each preparative gel and the region containing the cu-chtgn A cut out. As shown in Fig. 1, the preparative gels were cut at the midpoint of the CBP A-RBBR band and at the trailing edge of the Myo-RBBR band. The five gel slices, each 7-7.5 mm high, were then stacked in a gel tube having a slightly constricted bottom (Fig. 1, upper right). Electrophoresis buffer was used to remove trapped air bubbles from between the slices and along the wall of the tube. Protein recovery by electrophoresis into hydroxylapatite. As shown in Fig. 1 (lower right), an SDS-polyacrylamide gel plug (1.5 cm high) was cast in a second, slightly larger tube (1.35 cm i.d.). A 3 mm layer of G25 medium Sephadex was then placed on the gel plug. One gram of HA ’ The molecular weights of the proteins used are as follows (6,7): LDH = 36.000: CBP A = 34,400; cu-chtgn A = 25,741; Myo = 17.600; and cu-cht = 25.330 (A chain = 1240: B chain = 13,930; and C chain = 10.160).
368
ZIOLA
Ma$sr
0 l-l
Pmp&otive Q
X
---cut
Y
-cu
AND SCRABA
A
B C D
rlsllcrr Elrctropharorlr Buffor
I%
Hydronylapatitr
if
0
Sephadrx Layu Gal Plug
0
FIG. 1. (Left) Separation of a-chtgn A from a mixture of proteins by SDS-PAGE (diagrammatic). Gels of 1 x 6 cm (diam x length) were prepared and layered with protein mixtures as described in Methods. The direction of migration during electrophoresis was from top to bottom in the drawing (i.e., from 0 to @). The marker gel is shown as it appeared after electrophoresis; the coelectrophoresed preparative gel is shown as it would appear if stained with Coomassie blue. Slices were cut from the preparative gels as indicated. The protein bands (with their relative electrophoretic mobilities indicated in brackets) are as follows: A = LDH (0.77); B = a-chtgn A (1.00); C = a-cht B chain (1.33); D = cu-cht C chain (1.55); X = CBP A-RBBR (0.81); and Y = Myo-RBBR (1.24). (Right) Schematic representation of the apparatus used to recover the a-chtgn A from the preparative polyacrylamide gel slices. Dimensions and quantities of the components are given in the Methods section.
powder was suspended in electrophoresis buffer and fine material removed twice. The HA suspension was then thoroughly degassed and layered onto the Sephadex. The top and bottom tubes were brought together such that the elution surface of the top tube was just above the surface of the settled HA. After sealing the joint between the two tubes with parafilm to prevent evaporation of the buffer, electrophoresis was carried out at 25 mA for 8 hr. During this time the cY-chtgn A migrated from the gel slices into the HA. Using a Pasteur pipette with a 3-3.5 mm orifice, the HA was then carefully transferred to a glass column containing a small bed of G25 medium Sephadex. The HA was washed at 4-6 ml/hr with five column volumes of 0.12 M sodium phosphate (pH 6.4) containing 1 mM DTT and 0.1% SDS. The cu-chtgn A was then eluted by increasing the sodium phosphate concentration to 0.5 M.~ Protein recovery was monitored by measuring the absorbance (at 282 nm) of the eluate. Analysis of the recovered axhymotrypsinogen A. Following elution from the HA, the recovered a-chtgn A was divided into two portions. The first was dialyzed against 0.1% SDS in distilled water and ly* Moss and Rosenblum (3) have shown that SDS-complexed proteins are eluted from HA by 0.2-0.5 M sodium phosphate (pH 6.4), containing 0.1% SDS and 1 mM DTT.
PROTEIN
ISOLATlON
METHOD
369
ophilized. The resultant SDS-protein complex was dissolved at a final SDS concentration of 2% using 0.01 M sodium phosphate (pH 7.2) containing 5% P-mercaptoethanol and 10% glycerol. The solution was heated at 100°C for 5 min and the purity of the cY-chtgn A analyzed by electrophoresis of an aliquot in a 10% SDS-polyacrylamide gel (0.6 x 8 cm). For purposes of comparison, an aliquot of the starting mixture of proteins was concurrently electrophoresed in a separate gel (Fig. 2). The second portion of recovered cY-chtgn A was set to dialyze against 2000 vol of electrophoresis buffer. Dialyzed concurrently was a “standard” solution of a-chtgn A prepared by dissolving 1.5 mg of untreated protein in 2 ml of 0.01 M sodium phosphate (pH 7.2) containing 2% SDS and 5% P-mercaptoethanol, followed by heating at 100°C for 5 min. After dialysis at room temperature for 36 hr. the two dialyzates were adjusted to the same absorbance at 282 nm. The uv adsorption spectrum of each protein solution was then obtained using a Cary Model 15 recording spectrophotometer (Fig. 3). RESULTS
AND DISCUSSION
Recovery of cy-chtgn A was quantitative (93 + 5%, average of five determinations) following purification from a mixture of proteins by the method described. In addition, protein recoveries were greater than 85%
FIG. 2. Analytical SDS-PAGE of the a-chtgn A purified from a mixture of proteins by the method depicted in Fig. 1. For description of the lettering, see Fig. 1. The gel on the left shows the recovered ol-chtgn A: and the gel on the right, the starting protein mixture. Electrophoresis was for 4 hr at 5 mA/gel after which the gels were stained and destained according to the procedure of Weber and Osbom (6).
370
ZIOLA
AND SCRABA
0.8 -
0 240
270 300 WAVELENGTH (nm)
33q3
FIG. 3. The uv absorption spectrum of Lu-chtgn A after purification from a mixture of proteins by the method depicted in Fig. 1. Curve I corresponds to the recovered cu-chtgn A, and curve 2 corresponds to the original cy-chtgn A. See Methods for further details.
in single experiments involving bovine serum albumin, pepsin, and lysozyme. With respect to [14C]amino acid labeled Mengo virus (Y and y proteins, 90 to 95% recovery of radioactivity has been routinely achieved. Since the purified protein is eluted from the HA in concentrated form (total volume 3-4 ml), protein losses during subsequent reconcentration steps are minimized. The purity of the recovered cY-chtgn A is shown by Figs. 2 and 3. Analytical SDS-PAGE (Fig. 2) revealed that there was no contamination of the a-chtgn A by IDH or the B or C chains of a-cht. Comparison of the uv absorption spectrum of the recovered a-chtgn A with the spectrum of the original a-chtgn A (Fig. 3), indicated that there was very little contamination of the recovered protein by gel impurities which strongly absorb uv light of low wavelength. Any laboratory containing the materials necessary for analytical SDSPAGE can readily use the method described for protein purification. In doing so, two aspects must be considered. First, some means must be available for rapidly localizing the region of the preparative gels which contains the protein to be isolated. Concurrent electrophoresis of RBBRstained marker proteins is one method (Fig. 1, left). Should the protein to be purified have a high cysteine content, an alternative procedure is the reversible staining of the protein itself with the dye mercury orange (8,9). After the protein is eluted from the HA, the dye can be removed by treatment with acidic acetone. Acidic acetone not only regenerates the protein but also has the added advantage of precipitating the protein while leaving the SDS in solution (8,lO). In this regard, we have routinely recovered viral proteins from SDS-HA column eluates by acidic acetone precipitation (2). The second consideration is determining the minimum time necessary
PROTEIN
ISOLATION
METHOD
371
for electrophoresis of the protein from the gel slices into the HA (Fig. 1, right). In this regard, a RBBR-stained marker protein can be used once the components of the recovery apparatus and the electrophoretic conditions have been standardized. For example, with the protein recovery apparatus shown in Fig. 1 (and described above), it required 7 hr at 25 mA to electrophorese CBP A-RBBR from five gel slices into the HA. After electrophoresing for an additional 7 hr, the CBP A-RBBR was still completely contained within the HA, even though slow movement through the HA was evident. Consequently, for the recovery of cu-chtgn A (whose molecular weight is approximately 9000 less than that of CBP A). an electrophoresis clearing time of 8 hr was decided upon. If the protein to be recovered can be stained with mercury orange (see above), the electrophoresis of the protein from the gel slices into the HA can be followed directly. In conclusion, a procedure is described for the recovery of proteins following separation from contaminants by SDS-PAGE. The method can be undertaken using readily available and inexpensive materials. The new method overcomes the two main problems of preparative polyacrylamide gel electrophoresis systems: namely, recovery of the purified protein in diluted form and extensive contamination of the recovered protein by uv-absorbing gel impurities. ACKNOWLEDGMENTS The authors wish to express their thanks to Mr. Perry D’Obrenan for his assistance in preparing the figures herein and to Miss Cathy Hicks for her help with the photography involved. The studies were supported by Grant MA 4549 from the Medical Research Council of Canada. B. R. Z. is the recipient of a Science Scholarship from the National Research Council of Canada.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Chrambach, A., and Rodbard, D. (1971) Sciencel72,440. Ziola, B. R., and Scraba, D. G. (1975) Virology 64, 228. Moss. B., and Rosenblum, E. N. (1972) .I. Biol. Chem. 247, 5 194. Loening. U. E. (1967) Biochem. J. 102, 251. Griffith, 1. P. (1972) And. Biochem. 46, 402. Weber, K., and Osbom, M. (1969) J. Biol. Chem. 244,4406. Dunker, A. K., and Rueckert, R. R. (1969) J. Biol. C’hem. 244, 5074. Stoltzfus, C. M., and Rueckert, R. (1972) J. Yirol. 10, 347. Sakai, H. (19681 Anal. B&hem. 26, 269. Putnam, F. W. (1948)A&~zn. Protein Chem. 4, 79.
BIOCHEMISTRY
72, 366-371
(1976)
Recovery of SDS-Proteins from Polyacrylamide Gels by Electrophoresis into Hydroxylapatite BARRY R. ZIOLAANDDOUGLAS Department
of Biochemistry,
University of Alberta,
G. SCRABA Edmonton, Alberta
Received May 16, 1975; accepted November
T6G 2H7
19, 1975
A method is described which permits the purification of proteins in quantities necessary for physicochemical characterization. The protein to be purified is separated from contaminants by electrophoresis in a number of sodium dodecyl sulfate (SDS)-containing polyacrylamide disc gels. The region of each gel which contains the desired protein band is then cut out and the slices are stacked one above another in a tube. After the protein is electrophoresed from the gel slices into a bed of hydroxylapatite, it is eluted from the hydroxylapatite with 0.5 M sodium phosphate (pH 6.4) containing 0.1% SDS and 1 mM dithiothreitol. Protein recovery is greater than 90%.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) has gained wide acceptance as an analytical tool for assaying protein at progressive stages of purification. The application of SDS-PAGE to preparative procedures, however, has been hampered by dilution of the protein upon elution or extraction from gels and by contamination of the recovered protein by a gel impurity, which is believed to be linear polyacrylate (1). During the course of our studies on the Mengo encephalomyelitis virus capsid proteins (2), we occasionally encountered difficulty in separating two of the four virus structural polypeptides (i.e., the CYand y chains, of molecular weights 32,500 and 23,700, respectively) using the SDShydroxylapatite chromatography method of Moss and Rosenblum (3). As a result, we have developed a method for the quantitative recovery of these two proteins following their separation by SDS-PAGE. The method overcomes the protein dilution problem of most preparative electrophoresis systems; and in addition, uv absorption spectra of the recovered protein indicate that there is very little contamination by gel impurities. This preparative procedure should prove applicable to any polypeptide isolation where SDS-PAGE can be used for separation. For the purpose of illustration, a description is given of the isolation of a-chymotrypsinogen A (cu-chtgn A) from a mixture of proteins. METHODS Materials, Lactic dehydrogenase (LDH) and a-chtgn A were obtained from Sigma Chemical Co., carboxypeptidase A (CBP A) and a-chymo366 Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
PROTEIN
ISOLATION
METHOD
367
trypsin (a-cht) were from Worthington Biochemicals, and myoglobin (Myo) was from Schwarz/Mann Biochemicals.’ The dye Remazol Brilliant Blue R (RBBR) was purchased from Calbiochem. Acrylamide and N,N’methylenebisacrylamide were obtained from Eastman Organic Chemicals and recrystallized according to the procedure described by Loening (4). Dithiothreitol (DTT), technical grade SDS, and hydroxylapatite (HA) in powder form (Bio-Gel HTP) were purchased from Sigma Chemical Co., Matheson. Coleman, and Bell, and Bio-Rad Laboratories, respectively. All other chemicals were reagent grade. SDS-Polyacrylamide gel electrophoresis. All gels consisted of 10% acryalamide, 0.2% N,N’-methylenebisacrylamide, 0.1% SDS, and 0.1 M sodium phosphate (pH 7.2). Polymerization was catalyzed by N,N,N’N’tetramethylethylenediamine and ammonium persulfate, both at final concentrations of 0.075%. The gels were pre-electrophoresed for a minimum of 1 hr. LDH, cY-chtgn A, and a-cht were each dissolved in 0.01 M sodium phosphate (pH 7.2) containing 2% SDS and 10% glycerol. CBP A and Myo were stained by the protein-RBBR coupling procedure of Griffith (5) and similarly dissolved. A mixture containing 1.5 mg each of LDH and cY-chtgn A, and 3.0 mg of a-cht was made 5% with respect to P-mercaptoethanol (final volume, 770 ~1) and heated at 100°C for 5 min (under these conditions, a-cht dissociates to its constituent A, B, and C chains. Aliquots (150 ~1) were layered onto each of five SDS-polyacrylamide preparative gels (1 x 6 cm) directly beneath the electrophoresis buffer (0.1 M sodium phosphate, pH 7.2, containing 0.1% SDS). A 150 ~1 sample containing 300 pg of each of the prestained marker proteins, CPB A-RBBR and Myo-RBBR, was similarly prepared and layered onto a sixth gel. Electrophoresis was at 9-10 mA/gel for 10 hr. Following electrophoresis, the marker gel was aligned with each preparative gel and the region containing the cu-chtgn A cut out. As shown in Fig. 1, the preparative gels were cut at the midpoint of the CBP A-RBBR band and at the trailing edge of the Myo-RBBR band. The five gel slices, each 7-7.5 mm high, were then stacked in a gel tube having a slightly constricted bottom (Fig. 1, upper right). Electrophoresis buffer was used to remove trapped air bubbles from between the slices and along the wall of the tube. Protein recovery by electrophoresis into hydroxylapatite. As shown in Fig. 1 (lower right), an SDS-polyacrylamide gel plug (1.5 cm high) was cast in a second, slightly larger tube (1.35 cm i.d.). A 3 mm layer of G25 medium Sephadex was then placed on the gel plug. One gram of HA ’ The molecular weights of the proteins used are as follows (6,7): LDH = 36.000: CBP A = 34,400; cu-chtgn A = 25,741; Myo = 17.600; and cu-cht = 25.330 (A chain = 1240: B chain = 13,930; and C chain = 10.160).
368
ZIOLA
Ma$sr
0 l-l
Pmp&otive Q
X
---cut
Y
-cu
AND SCRABA
A
B C D
rlsllcrr Elrctropharorlr Buffor
I%
Hydronylapatitr
if
0
Sephadrx Layu Gal Plug
0
FIG. 1. (Left) Separation of a-chtgn A from a mixture of proteins by SDS-PAGE (diagrammatic). Gels of 1 x 6 cm (diam x length) were prepared and layered with protein mixtures as described in Methods. The direction of migration during electrophoresis was from top to bottom in the drawing (i.e., from 0 to @). The marker gel is shown as it appeared after electrophoresis; the coelectrophoresed preparative gel is shown as it would appear if stained with Coomassie blue. Slices were cut from the preparative gels as indicated. The protein bands (with their relative electrophoretic mobilities indicated in brackets) are as follows: A = LDH (0.77); B = a-chtgn A (1.00); C = a-cht B chain (1.33); D = cu-cht C chain (1.55); X = CBP A-RBBR (0.81); and Y = Myo-RBBR (1.24). (Right) Schematic representation of the apparatus used to recover the a-chtgn A from the preparative polyacrylamide gel slices. Dimensions and quantities of the components are given in the Methods section.
powder was suspended in electrophoresis buffer and fine material removed twice. The HA suspension was then thoroughly degassed and layered onto the Sephadex. The top and bottom tubes were brought together such that the elution surface of the top tube was just above the surface of the settled HA. After sealing the joint between the two tubes with parafilm to prevent evaporation of the buffer, electrophoresis was carried out at 25 mA for 8 hr. During this time the cY-chtgn A migrated from the gel slices into the HA. Using a Pasteur pipette with a 3-3.5 mm orifice, the HA was then carefully transferred to a glass column containing a small bed of G25 medium Sephadex. The HA was washed at 4-6 ml/hr with five column volumes of 0.12 M sodium phosphate (pH 6.4) containing 1 mM DTT and 0.1% SDS. The cu-chtgn A was then eluted by increasing the sodium phosphate concentration to 0.5 M.~ Protein recovery was monitored by measuring the absorbance (at 282 nm) of the eluate. Analysis of the recovered axhymotrypsinogen A. Following elution from the HA, the recovered a-chtgn A was divided into two portions. The first was dialyzed against 0.1% SDS in distilled water and ly* Moss and Rosenblum (3) have shown that SDS-complexed proteins are eluted from HA by 0.2-0.5 M sodium phosphate (pH 6.4), containing 0.1% SDS and 1 mM DTT.
PROTEIN
ISOLATlON
METHOD
369
ophilized. The resultant SDS-protein complex was dissolved at a final SDS concentration of 2% using 0.01 M sodium phosphate (pH 7.2) containing 5% P-mercaptoethanol and 10% glycerol. The solution was heated at 100°C for 5 min and the purity of the cY-chtgn A analyzed by electrophoresis of an aliquot in a 10% SDS-polyacrylamide gel (0.6 x 8 cm). For purposes of comparison, an aliquot of the starting mixture of proteins was concurrently electrophoresed in a separate gel (Fig. 2). The second portion of recovered cY-chtgn A was set to dialyze against 2000 vol of electrophoresis buffer. Dialyzed concurrently was a “standard” solution of a-chtgn A prepared by dissolving 1.5 mg of untreated protein in 2 ml of 0.01 M sodium phosphate (pH 7.2) containing 2% SDS and 5% P-mercaptoethanol, followed by heating at 100°C for 5 min. After dialysis at room temperature for 36 hr. the two dialyzates were adjusted to the same absorbance at 282 nm. The uv adsorption spectrum of each protein solution was then obtained using a Cary Model 15 recording spectrophotometer (Fig. 3). RESULTS
AND DISCUSSION
Recovery of cy-chtgn A was quantitative (93 + 5%, average of five determinations) following purification from a mixture of proteins by the method described. In addition, protein recoveries were greater than 85%
FIG. 2. Analytical SDS-PAGE of the a-chtgn A purified from a mixture of proteins by the method depicted in Fig. 1. For description of the lettering, see Fig. 1. The gel on the left shows the recovered ol-chtgn A: and the gel on the right, the starting protein mixture. Electrophoresis was for 4 hr at 5 mA/gel after which the gels were stained and destained according to the procedure of Weber and Osbom (6).
370
ZIOLA
AND SCRABA
0.8 -
0 240
270 300 WAVELENGTH (nm)
33q3
FIG. 3. The uv absorption spectrum of Lu-chtgn A after purification from a mixture of proteins by the method depicted in Fig. 1. Curve I corresponds to the recovered cu-chtgn A, and curve 2 corresponds to the original cy-chtgn A. See Methods for further details.
in single experiments involving bovine serum albumin, pepsin, and lysozyme. With respect to [14C]amino acid labeled Mengo virus (Y and y proteins, 90 to 95% recovery of radioactivity has been routinely achieved. Since the purified protein is eluted from the HA in concentrated form (total volume 3-4 ml), protein losses during subsequent reconcentration steps are minimized. The purity of the recovered cY-chtgn A is shown by Figs. 2 and 3. Analytical SDS-PAGE (Fig. 2) revealed that there was no contamination of the a-chtgn A by IDH or the B or C chains of a-cht. Comparison of the uv absorption spectrum of the recovered a-chtgn A with the spectrum of the original a-chtgn A (Fig. 3), indicated that there was very little contamination of the recovered protein by gel impurities which strongly absorb uv light of low wavelength. Any laboratory containing the materials necessary for analytical SDSPAGE can readily use the method described for protein purification. In doing so, two aspects must be considered. First, some means must be available for rapidly localizing the region of the preparative gels which contains the protein to be isolated. Concurrent electrophoresis of RBBRstained marker proteins is one method (Fig. 1, left). Should the protein to be purified have a high cysteine content, an alternative procedure is the reversible staining of the protein itself with the dye mercury orange (8,9). After the protein is eluted from the HA, the dye can be removed by treatment with acidic acetone. Acidic acetone not only regenerates the protein but also has the added advantage of precipitating the protein while leaving the SDS in solution (8,lO). In this regard, we have routinely recovered viral proteins from SDS-HA column eluates by acidic acetone precipitation (2). The second consideration is determining the minimum time necessary
PROTEIN
ISOLATION
METHOD
371
for electrophoresis of the protein from the gel slices into the HA (Fig. 1, right). In this regard, a RBBR-stained marker protein can be used once the components of the recovery apparatus and the electrophoretic conditions have been standardized. For example, with the protein recovery apparatus shown in Fig. 1 (and described above), it required 7 hr at 25 mA to electrophorese CBP A-RBBR from five gel slices into the HA. After electrophoresing for an additional 7 hr, the CBP A-RBBR was still completely contained within the HA, even though slow movement through the HA was evident. Consequently, for the recovery of cu-chtgn A (whose molecular weight is approximately 9000 less than that of CBP A). an electrophoresis clearing time of 8 hr was decided upon. If the protein to be recovered can be stained with mercury orange (see above), the electrophoresis of the protein from the gel slices into the HA can be followed directly. In conclusion, a procedure is described for the recovery of proteins following separation from contaminants by SDS-PAGE. The method can be undertaken using readily available and inexpensive materials. The new method overcomes the two main problems of preparative polyacrylamide gel electrophoresis systems: namely, recovery of the purified protein in diluted form and extensive contamination of the recovered protein by uv-absorbing gel impurities. ACKNOWLEDGMENTS The authors wish to express their thanks to Mr. Perry D’Obrenan for his assistance in preparing the figures herein and to Miss Cathy Hicks for her help with the photography involved. The studies were supported by Grant MA 4549 from the Medical Research Council of Canada. B. R. Z. is the recipient of a Science Scholarship from the National Research Council of Canada.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Chrambach, A., and Rodbard, D. (1971) Sciencel72,440. Ziola, B. R., and Scraba, D. G. (1975) Virology 64, 228. Moss. B., and Rosenblum, E. N. (1972) .I. Biol. Chem. 247, 5 194. Loening. U. E. (1967) Biochem. J. 102, 251. Griffith, 1. P. (1972) And. Biochem. 46, 402. Weber, K., and Osbom, M. (1969) J. Biol. Chem. 244,4406. Dunker, A. K., and Rueckert, R. R. (1969) J. Biol. C’hem. 244, 5074. Stoltzfus, C. M., and Rueckert, R. (1972) J. Yirol. 10, 347. Sakai, H. (19681 Anal. B&hem. 26, 269. Putnam, F. W. (1948)A&~zn. Protein Chem. 4, 79.
