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Removal of Sodium Dodecyl Sulphate from Proteins Isolated by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis Batia Kaplant and Mordechai Pras H e l l e r Institute of Medical Research, Chaim Sheba Medical Center, Sackler Medical School, Tel-Aviv University, Tel-Aviv. Israel
A procedure is described for the removal of sodium dodecyl sulphate (SDS) from proteins isolated by SDSpolyacrylamide gel electrophoresis (SDS-PAGE). The proteins separated by SDS-PAGE were stained with Coomassie Blue and extracted with Tris-HCI buffer containing SDS. The obtained extracts were subjected to gel permeation chromatography in an acidic aqueous acetonitrile solution. The procedure allows purification of the isolated proteins not only from SDS, but also from Coomassie Blue, buffer salts and other small molecular weight contaminants.
INTRODUCTION Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is one of the most effective methods in protein separation. This method is widely used as an analytical tool to determine the purity and molecular weight of proteins. However, the preparative application of SDS-PAGE is often complicated by the contamination of the proteins, recovered from the gel, with SDS, acrylamide and other substances (Ilunkapiller et al, 1983). Recently, we have developed a method for SDS removal from SDS + protein complexes by using gel permeation chromatography in organic solvent (Kaplan and Pras, 1987a). In the present study this method was applied to the purification of proteins recovered from the SDS gels.
The extracts obtained (2.2-2.8 mL) were lyophilized and redissolved in 0.6-0.9 mL of aqueous 0.1% trifluoroacetic acid (TFA) (Sigma). The same volume of the solution containing 0.1% TFA in acetonitrile (Bio-Lab, Jerusalem, Israel) was added gradually with mixing. The soluble samples obtained were separated on a Fractogel TSK HW-40 (F) (Merck),
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SDS-PAGE was performed using 17% polyacrylamide slab gel (Laemmli, 1970), 1.5 mm thick, 5 mm wide sample wells. Acrylamide was purchased from Sigma (St. Louis, MO, USA). Bovine serum albumin (BSA), ovalbumin, p-lactoglobulin, lysozyme (Sigma) and amyloid protein A (obtained as reported earlier by Pras et al. (1980)) were used in our experiments. The mixture of these proteins (90-350 kg of each protein) was loaded into several wells (25-30 pL per well). Proteins were stained with Coomassie Blue (Sigma). The bands of each separated protein were cut out and subjected to the extraction procedure using 0.05 mol/L Tris-HCl buffer (pH 7.0) containing 0.1% SDS (BDH, Poole, UK), 0.1 mmol/L EDTA (Merck, Darmstadt, FRG), 5 mmol/ L dithioerythriol (Sigma), and 0.2 mol/L NaCl (Hager and Burges, 1980; Kaplan and Pras, 1987b). The extraction procedure was repeated three times. $ Author to whom correspondence should be addressed at Heller Institute of Medical Research, Sheba Medical Center, 52621 TelHashomer, Israel.
2m
D 111
4
0.4
0.2
0.c
I
10
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Figure 1. Removal of SDS and Coomassie Blue from proteins (extracted from SDS gels) by gel permeation Chromatography in aqueous 50% acetonitrile containing 0.1% TFA. The proteins (180360 pg) were run by SDS-PAGE, stained and extracted as described in Experimental. The extracted materials containing P-lactoglobulin were chromato(A), lysozyme (B). BSA (C) and ovalbumin (0) graphed on a Fractogel TSK HW-40 (F) column, 2 0 5 ~ 1 4 m m .ID. Fractions of 1.1 mL were collected and checked for SDS (x, peak Ill),stain (0,peak II) and protein (0,peak I). For the determination of protein and SDS in fractions 1-10 the samples were 6-9-fold concentrated (by lyophilization) prior to their analysis.
CCC-0269-3879/90/0089-0090 $1.00 @ Heyden & Son Limited, 1990
BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 2, 1990 89
B. KAPLAN AND M. PRAS
column 205 x 14 mm ID, in aqueous 50% acetonitrile containing 0.1% TFA. Elution of proteins was checked by the method of Lowry etal. (1951), SDS-using basic fuchsin (BDH) (Waite and Wang, 1976). For evaluation of the efficiency of this procedure the eluted proteins were run by SDS-PAGE and stained with Coomassie Blue. The amount of the bound stain was referenced to that obtained for a known amount of protein analysed in the same run (Kaplan and Pras, 1987b).
RESULTS The proteins were run by SDS-PAGE, stained and extracted with the SDS-containing buffer. The extracts, containing protein, stain, buffer salts and gel contaminants, were concentrated and acidified, as described in Experimental. The samples obtained (0.6-0.9 mL) were diluted twice with 0.1% TFA in acetonitrile prior to their application on the Fractogel column. Typical chromatographic patterns of these samples are shown in Fig. 1. Good separation of proteins (Vo, peak I) from SDS (Peak 11) and Coomassie Blue (Peak 111) was obtained. All the proteins extracted from the SDS gels and afterwards eluted from the Fractogel column were free from SDS (
Removal of Sodium Dodecyl Sulphate from Proteins Isolated by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis Batia Kaplant and Mordechai Pras H e l l e r Institute of Medical Research, Chaim Sheba Medical Center, Sackler Medical School, Tel-Aviv University, Tel-Aviv. Israel
A procedure is described for the removal of sodium dodecyl sulphate (SDS) from proteins isolated by SDSpolyacrylamide gel electrophoresis (SDS-PAGE). The proteins separated by SDS-PAGE were stained with Coomassie Blue and extracted with Tris-HCI buffer containing SDS. The obtained extracts were subjected to gel permeation chromatography in an acidic aqueous acetonitrile solution. The procedure allows purification of the isolated proteins not only from SDS, but also from Coomassie Blue, buffer salts and other small molecular weight contaminants.
INTRODUCTION Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is one of the most effective methods in protein separation. This method is widely used as an analytical tool to determine the purity and molecular weight of proteins. However, the preparative application of SDS-PAGE is often complicated by the contamination of the proteins, recovered from the gel, with SDS, acrylamide and other substances (Ilunkapiller et al, 1983). Recently, we have developed a method for SDS removal from SDS + protein complexes by using gel permeation chromatography in organic solvent (Kaplan and Pras, 1987a). In the present study this method was applied to the purification of proteins recovered from the SDS gels.
The extracts obtained (2.2-2.8 mL) were lyophilized and redissolved in 0.6-0.9 mL of aqueous 0.1% trifluoroacetic acid (TFA) (Sigma). The same volume of the solution containing 0.1% TFA in acetonitrile (Bio-Lab, Jerusalem, Israel) was added gradually with mixing. The soluble samples obtained were separated on a Fractogel TSK HW-40 (F) (Merck),
B 111
0.4
i-'
Q2
E
5 0.0 .
Y ~
z u
EXPERIMENTAL
C
4
m
SDS-PAGE was performed using 17% polyacrylamide slab gel (Laemmli, 1970), 1.5 mm thick, 5 mm wide sample wells. Acrylamide was purchased from Sigma (St. Louis, MO, USA). Bovine serum albumin (BSA), ovalbumin, p-lactoglobulin, lysozyme (Sigma) and amyloid protein A (obtained as reported earlier by Pras et al. (1980)) were used in our experiments. The mixture of these proteins (90-350 kg of each protein) was loaded into several wells (25-30 pL per well). Proteins were stained with Coomassie Blue (Sigma). The bands of each separated protein were cut out and subjected to the extraction procedure using 0.05 mol/L Tris-HCl buffer (pH 7.0) containing 0.1% SDS (BDH, Poole, UK), 0.1 mmol/L EDTA (Merck, Darmstadt, FRG), 5 mmol/ L dithioerythriol (Sigma), and 0.2 mol/L NaCl (Hager and Burges, 1980; Kaplan and Pras, 1987b). The extraction procedure was repeated three times. $ Author to whom correspondence should be addressed at Heller Institute of Medical Research, Sheba Medical Center, 52621 TelHashomer, Israel.
2m
D 111
4
0.4
0.2
0.c
I
10
T O - - - - 20 FRACTION NUMBER
__
70
Figure 1. Removal of SDS and Coomassie Blue from proteins (extracted from SDS gels) by gel permeation Chromatography in aqueous 50% acetonitrile containing 0.1% TFA. The proteins (180360 pg) were run by SDS-PAGE, stained and extracted as described in Experimental. The extracted materials containing P-lactoglobulin were chromato(A), lysozyme (B). BSA (C) and ovalbumin (0) graphed on a Fractogel TSK HW-40 (F) column, 2 0 5 ~ 1 4 m m .ID. Fractions of 1.1 mL were collected and checked for SDS (x, peak Ill),stain (0,peak II) and protein (0,peak I). For the determination of protein and SDS in fractions 1-10 the samples were 6-9-fold concentrated (by lyophilization) prior to their analysis.
CCC-0269-3879/90/0089-0090 $1.00 @ Heyden & Son Limited, 1990
BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 2, 1990 89
B. KAPLAN AND M. PRAS
column 205 x 14 mm ID, in aqueous 50% acetonitrile containing 0.1% TFA. Elution of proteins was checked by the method of Lowry etal. (1951), SDS-using basic fuchsin (BDH) (Waite and Wang, 1976). For evaluation of the efficiency of this procedure the eluted proteins were run by SDS-PAGE and stained with Coomassie Blue. The amount of the bound stain was referenced to that obtained for a known amount of protein analysed in the same run (Kaplan and Pras, 1987b).
RESULTS The proteins were run by SDS-PAGE, stained and extracted with the SDS-containing buffer. The extracts, containing protein, stain, buffer salts and gel contaminants, were concentrated and acidified, as described in Experimental. The samples obtained (0.6-0.9 mL) were diluted twice with 0.1% TFA in acetonitrile prior to their application on the Fractogel column. Typical chromatographic patterns of these samples are shown in Fig. 1. Good separation of proteins (Vo, peak I) from SDS (Peak 11) and Coomassie Blue (Peak 111) was obtained. All the proteins extracted from the SDS gels and afterwards eluted from the Fractogel column were free from SDS (
