ANALYTICAL BIOCHEMISTRY 70, 3 2 - 3 8
(1976)
A Highly Crosslinked, Transparent Polyacrylamide Gel with Improved Mechanical Stability for Use in Isoelectric Focusing and Isotachophoresis GERHARD BAUMANN AND ANDREAS CHRAMBACH Reproduction Research Branch, National Institute for Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014
Received December 5, 1974; accepted August 14, 1975
Polyacrylamide gels highly (10-50%) crosslinked with N,N'-methylenebisacrylamide (BIS) provide large pore sizes but are mechanically labile. By substituting N,N'-diallyltartardiamide (DATD) for BIS, mechanical stability and firm adherence to glass walls is conferred to these gels, with only a minor decrease in pore size compared to highly BIS-crosslinked gels. The highly DATD-crosslinked gels have the further advantage of being transparent. It appears that highly DATD-crosslinked gels combining large pore size with mechanical stability are superior to presently used gel types in isoelectric focusing on polyacrylamide gel and isotachophoresis on polyacrylamide gel.
A stacking gel with relatively large effective pore size, designed to minimize retardation of proteins, was introduced by Ornstein and Davis in 1960 (1,2). Their choice of gel concentration proved extraordinarily suitable in the light of later evidence: a 3.125% T, 20% C (BIS) 1 gel has a pore size large enough to allow the relatively unrestricted passage of compounds with a molecular weight of several million (3). T h e soft consistency and poor wall adherence o f these stacking gels is somewhat of a disadvantage, but in ordinary polyacrylamide-gel electrophoresis ( P A G E ) in multiphasic buffer systems (4) the stacking gel is supported by a sturdier resolving gel. T h e mechanical stability of these gels becomes a limiting factor in two applications where no supporting gel or other stabilizing medium is used: (i) isoelectric focusing o n polyacrylamide gel ( I F P A ) and (ii) isotachophoresis on polyacrylamide gel ( I T P P A ) . In I F P A the problem of wall adherence is aggravated by p H - d e p e n d e n t swelling and constriction of the gel, leading to convection and current flow along the walls (5). In I T P P A , heating across the stacked, highly concentrated protein zone can lead to severe distortion, rupture, and wall separation o f 1 BIS, N,N'-methylenebisacrylamide; N, N ,N ', N '-tetramethylethylenediamine.
DATD, N,N'-diallyltartardiamide; Temed,
32 Copyright © 1976 by Academic Press, Inc All rights of reproduction m any form reserved.
MECHANICALLYSTABLE STACKING GELS
33
"nonrestrictive ''2 gels (6). The mechanical properties of gels can be improved by increasing the total gel concentration and/or decreasing the percentage of crosslinking, but both of these maneuvers are accompanied by a decrease in pore size (3,7). This can be tolerated if the compound under investigation is shown to be unrestricted (stacked) in such a gel by a preliminary Ferguson-plot analysis (5,8). This report describes an attempt to arrive at a universally applicable nonrestrictive gel with improved mechanical properties.
MATERIALS AND METHODS Proteins. Ovine prolactin (oPRL; molecular weight (MW 23,000) was a gift from the National Pituitary Agency. Bovine serum albumin (BSA; MW 67,000) and rabbit phosphorylase B (MW 100,000) were purchased from Sigma Chemical Company, St. Louis, Missouri. Rabbit aldolase (MW 150,000) was purchased from Boehringer Company, Mannheim, Germany. Ovalbumin (MW 44,000) and catalase (MW 240,000) were purchased from Worthington Biochemical Corporation, Freehold, New Jersey. Equine ferritin (MW 470,000) was purchased from Grand Island Biological Company. Human immunoglobulin M (IgM; MW 1 million) was a gift from Dr. P. H. Plotz, N I A M D D , and low density lipoprotein (LDL; MW 3.2 million) was a gift from Dr. H. R. Sloan, N H L I , NIH. Reagents. Acrylamide (Cat. No. 0019) and BIS (Cat. No. 0719) were purchased from Polysciences Inc., Warrington, Pennsylvania. D A T D 1 (Cat. No. BO-71005) was purchased from General Biochemicals, Chagrin Falls, Ohio, or (Cat. No. 161-0620) from Bio-Rad, Richmond, California. All other chemicals were reagent grade. PAGE, IFPA, and ITPPA. Stacking gels of 2.5-30% T, 15-30% C (BIS or DATD) were used in multiphasic buffer system 2950 (operative pl-i, 10.4) (8,9, Appendixl). These gels are designated as "highly crosslinked." For comparison, 4% C (BIS) gels ranging from 3 to 25% T were used in the same buffer system. Initiator mixtures consisted of 5 × 10-4% riboflavin, 0.015% potassium persulfate, 1 /zl of Temedl/ml at 0°C. The polymerization procedure and verification of stacking was carried out as described (7,8). Preparative ITPPA was performed in 5% T, 15% C (DATD) gels in preparative apparatus C and D as reported elsewhere (6,10). I F P A was performed in the same gel types as described (11). Gels containing synthetic ampholytes were stained according to the procedure of Reisner et al. (12). 2 The term "nonrestrictive,"as used in the context of this report, should not be perceived in absolute terms but relative to the propagationrate of the movingboundary(19). It is emphasized that gels are necessarily restrictive to even small molecules, such as buffer ions (20), urea, and D20 (13,2l).
34
BAUMANN
AND CHRAMBACH
RESULTS
The mechanical stability and wall adherence of highly crosslinked gels is substantially increased when BIS is substituted by D A T D as the crosslinking agent. In IFPA, gels highly crosslinked with BIS had a tendency to fall out of the glass tubes, while gels crosslinked with DATD showed no vertical displacement under identical conditions. In addition, pH-dependent shrinking and swelling of the gels did not lead to visible separation from the glass wall in DATD-crosslinked gels, but wall separation was always noted in gels highly crosslinked with BIS. The most impressive evidence for these improved mechanical properties is apparent in preparative ITPPA (6): Large 50-200-ml gel blocks could be maintained for several days without noticeable distortion of the surface or evidence of wall separation. An added advantage is the complete transparency of highly DATD-crosslinked gels in contrast to the white opacity of highly BIS-crosslinked gels. These advantages of DATD-crosslinked gels are in part offset by an increase in molecular sieving compared to gels highly crosslinked with BIS. The relative magnitude of molecular sieving effects in highly DATDcrosslinked gels, highly crosslinked-BIS gels, and conventional (2-5% crosslinked) BIS gels was evaluated by measurement of Rs in P A G E at pH 10.4, using stacking gels of varying concentrations. The results for a series of proteins with molecular weights ranging from 23,000 to 3.2 million are given in Tables 1 and 2. The retardation coefficient, Ka, is the measure of pore size in gel electrophoresis under any given set of conditions of pH, temperature, and propagation rate of the reference moving boundary (13). KR values are smaller in 15% DATD-crosslinked gels than in 4% BIS-crosslinked gels, indicating that the latter are more TABLEI RETARDATION COEFFICIENTS (KR) OF STANDARD PROTEINS IN 15% DATD-CRosSLINKED AND 4% BIS-CRosSLINKED GELS (BUFFER SYSTEM NO. 2950, PHASE ZETA, PH 10.4, 0°C) Kn Protein oPRL Ovalbumin BSA Phosphorylase B Aldolase Catalase Ferritin IgM LDL
Molecular Weight 2.3 4.4 6.7 1.0 1.5 2.4 4.7 1.0 3.2
x x x x x x x x x
104 104 104 l0 s 105 105 l0 s 106 10n
15% C ( D A T D )
4% C (BIS)
0.018 0.020 0.036 0.045 0.061 0.069 0.105 0.219 0.234
0.047 0.060 0.106 0.106 0.147 0.146 0.365 0.377 1.217
MECHANICALLY STABLE STACKING GELS
35
TABLE 2 MAXIMALGEL CONCENTRATIONSOF 15% DATD-CROSSLINKEDGELS WHICH PROVIDEAN Re = 1.00 AT PH 10.4 (LOWER STACKINGLIMIT, RM (1, ZETA)= 0.046) FOR THE PROTEINS SHOWNa Gel Concentration (% T) Protein oPRL Ovalbumin BSA Phosphorylase B Aldolase Catalase Ferritin IgM LDL
MolecularWeight 2.3 4.4 6.7 1.0 1.5 2.4 4.7 1.0 3.2
× × × x x × × × ×
104 104 10a 10~ 105 105 105 106 106
15% C (DATD)
4% C (BIS)
20 18 15 10 9 7.5 6 5 3.5
11 10 10 7.5 6 5 5 3 < 3
a An Rs = 1.00 of a protein indicates that the protein is stacked within a moving boundary migrating at a particular velocity. When this velocity is low, such as in the buffer system selected here, proteins are more likely to exhibit an Rs = 1.00. Although under these circumstances the protein has a mobility in the range of the buffer constituents, an Rs of 1.00 does not necessarily indicate "absence" of molecular sieving effects. An increase of the velocity of the moving boundary [RM (1, ZETA)] by using another buffer system would eventually lead to unstacking of the protein at the same gel concentration. Therefore, the "Re = 1.00 test" represents only a rough guide for "nonrestrictiveness." An absolute measure for molecular sieving effects can only be obtained by the more laborious procedure of determining KB.
r e s t r i c t i v e . A s a p r a c t i c a l g u i d e , t h e m a x i m a l gel c o n c e n t r a t i o n s t h a t a r e n o n r e s t r i c t i v e to a s e r i e s o f p r o t e i n s w i t h d i f f e r e n t m o l e c u l a r w e i g h t s a r e s h o w n in T a b l e 2. A c o m p a r i s o n b e t w e e n t h e p o r e s i z e s o f 15% C D A T D g e l s a n d 15% C B I S gels w a s m a d e using L D L . L D L m i g r a t e d w i t h R s = 1.00 in 15% B I S - c r o s s l i n k e d gels up to a gel c o n c e n t r a t i o n o f 9 % T . W h i l e a n i n c r e a s i n g d e g r e e o f c r o s s l i n k i n g up to 50% C ( B I S ) h a s b e e n r e p o r t e d to r e s u l t in p r o g r e s s i v e l y l a r g e r p o r e size (3), t h e p o r e size o f D A T D - c r o s s l i n k e d gels a p p e a r e d t o r e m a i n a p p r o x i m a t e l y c o n s t a n t f r o m 15% t o 30% C. Figure 1 illustrates the advantages of highly DATD-crosslinked gels in I F P A as c o m p a r e d to a c o n v e n t i o n a l 4 % T , 4% C ( B I S ) gel. I t is c l e a r l y a p p a r e n t t h a t t h e p r o t e i n s a r e m o r e s e v e r e l y r e t a r d e d in t h e i r m i g r a t i o n t o w a r d t h e i s o e l e c t r i c p o i n t in t h e c o n v e n t i o n a l 4 % B I S - c r o s s l i n k e d gel t h a n in t h e h i g h l y c r o s s l i n k e d D A T D gel. L D L d i d n o t e n t e r t h e 4 % T , 4 % C ( B I S ) g e l e x c e p t for a v e r y m i n o r c o m p o n e n t . A n a t t e m p t w a s m a d e to c o m b i n e t h e a d v a n t a g e s o f B I S gels a n d D A T D gels ( o p e n p o r e size a n d m e c h a n i c a l s t a b i l i t y , r e s p e c t i v e l y ) b y
36
BAUMANN AND CHRAMBACH
$-'?/'
A.
B.
A.
B.
Fro. 1. Protein patterns after 30 min of I F P A (ampholyte pI-range 3.5-10, 0°C) at 200 V. The two gels on the left side contain IgM, those on the right LDL. The proteins were applied at the top. The anode is placed at the bottom. (A) 4% T, 4% C (BIS) gel; (B) 5% T, 15% C (DATD) gel. Arrows indicate migration distances/30 rain for representative bands.
using mixtures of the two crosslinking agents in various proportions. It was found that the admixture of 0.5-3% D A T D to B1S (w/w) resulted in improved wall adherence without significant decrease in pore size. Opacity of these gels decreased with increasing percentage of DATD. LDL remained unrestricted in such gels up to a gel concentration of 9% T, 15% C. Higher percentages of DATD, however, resulted in gel properties much like those of pure DATD-crosslinked gels. DISCUSSION The need for a nonrestrictive gel in stacking, ITPPA, and IFPA derives from the fact that significant retardation of the compound under investigation results in unstacking in the first two cases and, in the latter, inability to reach the isoelectric position during the life-span of the pH gradient. The previous finding that the effective pore size of polyacrylamide gels could be increased significantly by increasing the degree of crosslinking seemed to provide a tool for making such nonrestrictive gels (3). However, the mechanical properties of BIS-crosslinked gels in the range of interest (10-50% C) deteriorate with increasing degree of crosslinking. Such gels become increasingly brittle and exhibit decreasing wall adherence. They are also opaque and adsorb dyes used for the staining of protein bands. Previous experience with polyacrylamide gels crosslinked with D A T D and ethylene-diacrylate for purposes of easy solubilization (14,15)
M E C H A N I C A L L Y STABLE S T A C K I N G GELS
37
prompted us to investigate the applicability of highly crosslinked D A T D gels to ITPPA and IFPA. It appears from the present data that highly DATD-crosslinked gels (e.g., a 5% T, 15% C ( D A T D ) gel) provide desirable mechanical stability, wall adherence, and a pore size sufficiently open to permit the unrestricted passage of compounds not exceeding a molecular weight of 0.5 million. Such gels are not brittle, support their own weight even in large blocks, and can be sliced with adequate precision (16). The availability of a generally applicable IFPA and ITPPA gel obviates the need to investigate, in each particular instance, the maximal gel concentration that is nonrestrictive to the compound of interest. The 5% T, 15% C (DATD) gel should replace previously used mechanically stable, but more restrictive, gels both in IFPA (17) and in ITPPA (18), which may result in unstacking or inability to reach the isoelectric position. ACKNOWLEDGMENT This work was supported in part by "Stiftung fiir Biologisch-Medizinische Stipendien," Switzerland, APPENDIX 1 MULTIPHASIC BUFFER SYSTEM No. 2950
Upper buffer
Stacking gel buffer
~/-Aminobutyric acid 2-Amino-2-methylpropane-1,3-diol HzO
4.12 g 4.34 g to 1000 ml
1 N H2SO4 H20
21.3 ml 2.30 g to 100 ml
1 N HCI 2-Amino-2-methylpropane- 1,3-diol HzO
50.0 ml 6.57 g to 1000 ml
2-Amino-2-methylpropane-1,3-diol Lower buffer
REFERENCES 1. 2. 3. 4. 5.
Ornstein, L. L. (1964)Ann. N . Y . Acad. Sci. 121, 321. Davis, B. J. (1964)Ann. N . Y . Acad. Sci. 121, 404. Rodbard, D., Levitov, C., and Chrambach, A. (1972) Separ. Sci. 7, 705. Chrambach, A., and Rodbard, D. (1971)Science 172, 440. Chrambach, A., Doerr, P., Finlayson, G. R., Miles, L. E. M., Sherins, R., and Rodbard, D. (1973)Ann. N . Y . Acad. Sci. 209, 44. 6. Baumann, G., and Chrambach, A., Proc. Nat. Acad. Sci. USA, in press. 7. Rodbard, D., and Chrambach, A. (1971) Anal. Biochem. 40, 95. 8. Rodbard, D., and Chrambach, A. (1974) in Electrophoresis and Isoelectric Focusing on Polyacrylamide Gel (Allen, R. C., and Maurer, H. R., eds.), p. 62, Walter de Gruyter, Berlin.
38
BAUMANN AND CHRAMBACH
9. Jovin, T. M. (1973)Biochemist~ 12, 871. 10. Kapadia, G., and Chrambach, A. (1972)Anal. Biochem. 48, 90; Appendix III; Preparative PAGE Procedure: Apparatus C and D. 11. Baumann, G., and Chrambach, A. (1975) in Progress in Isoelectric Focusing and Isotachophoresis (Righetti, P. G., ed.), Elsevier, Excerpta Medica, North-Holland, Assoc. Sci. Publ., Amsterdam, in press. 12. Reisner, A. H., Nemes, P., and Bucholtz, C. (1975)Anal. Biochem. 64, 509. 13. Rodbard, D., and Chrambach, A. (1970) Proc. Nat. Acad. Sci. USA 65, 970. 14. Anker, H. S. (1970) F E B S Lett. 7, 293. 15. Choules, G. L., and Zimm, B. H. (1965)Anal. Biochem. 13, 336. 16. Peterson, J. I., Tipton, H. W., and Chrambach, A. (1974)Anal. Biochem. 62, 274. 17. Righetti, P. J., and Drysdale J. W. (1974) J. Chromatogr. 98, 271. 18. Svendsen, P. J., and Rose. C. (1970) Sci. Tools 17, 13. 19. Chrambach, A., and Baumann, G. (1975) in Isoelectric Focusing (Catsimpoolas, N., ed.), Academic Press, New York, in press. 20. Chrambach, A., Hearing, E., Lunney, J., and Rodbard, D., (1972)Separ. Sci. 7 725. 21. White, M. L., and Dorion, G. H. (1961) J. Polym. Sci. 55, 731.
(1976)
A Highly Crosslinked, Transparent Polyacrylamide Gel with Improved Mechanical Stability for Use in Isoelectric Focusing and Isotachophoresis GERHARD BAUMANN AND ANDREAS CHRAMBACH Reproduction Research Branch, National Institute for Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014
Received December 5, 1974; accepted August 14, 1975
Polyacrylamide gels highly (10-50%) crosslinked with N,N'-methylenebisacrylamide (BIS) provide large pore sizes but are mechanically labile. By substituting N,N'-diallyltartardiamide (DATD) for BIS, mechanical stability and firm adherence to glass walls is conferred to these gels, with only a minor decrease in pore size compared to highly BIS-crosslinked gels. The highly DATD-crosslinked gels have the further advantage of being transparent. It appears that highly DATD-crosslinked gels combining large pore size with mechanical stability are superior to presently used gel types in isoelectric focusing on polyacrylamide gel and isotachophoresis on polyacrylamide gel.
A stacking gel with relatively large effective pore size, designed to minimize retardation of proteins, was introduced by Ornstein and Davis in 1960 (1,2). Their choice of gel concentration proved extraordinarily suitable in the light of later evidence: a 3.125% T, 20% C (BIS) 1 gel has a pore size large enough to allow the relatively unrestricted passage of compounds with a molecular weight of several million (3). T h e soft consistency and poor wall adherence o f these stacking gels is somewhat of a disadvantage, but in ordinary polyacrylamide-gel electrophoresis ( P A G E ) in multiphasic buffer systems (4) the stacking gel is supported by a sturdier resolving gel. T h e mechanical stability of these gels becomes a limiting factor in two applications where no supporting gel or other stabilizing medium is used: (i) isoelectric focusing o n polyacrylamide gel ( I F P A ) and (ii) isotachophoresis on polyacrylamide gel ( I T P P A ) . In I F P A the problem of wall adherence is aggravated by p H - d e p e n d e n t swelling and constriction of the gel, leading to convection and current flow along the walls (5). In I T P P A , heating across the stacked, highly concentrated protein zone can lead to severe distortion, rupture, and wall separation o f 1 BIS, N,N'-methylenebisacrylamide; N, N ,N ', N '-tetramethylethylenediamine.
DATD, N,N'-diallyltartardiamide; Temed,
32 Copyright © 1976 by Academic Press, Inc All rights of reproduction m any form reserved.
MECHANICALLYSTABLE STACKING GELS
33
"nonrestrictive ''2 gels (6). The mechanical properties of gels can be improved by increasing the total gel concentration and/or decreasing the percentage of crosslinking, but both of these maneuvers are accompanied by a decrease in pore size (3,7). This can be tolerated if the compound under investigation is shown to be unrestricted (stacked) in such a gel by a preliminary Ferguson-plot analysis (5,8). This report describes an attempt to arrive at a universally applicable nonrestrictive gel with improved mechanical properties.
MATERIALS AND METHODS Proteins. Ovine prolactin (oPRL; molecular weight (MW 23,000) was a gift from the National Pituitary Agency. Bovine serum albumin (BSA; MW 67,000) and rabbit phosphorylase B (MW 100,000) were purchased from Sigma Chemical Company, St. Louis, Missouri. Rabbit aldolase (MW 150,000) was purchased from Boehringer Company, Mannheim, Germany. Ovalbumin (MW 44,000) and catalase (MW 240,000) were purchased from Worthington Biochemical Corporation, Freehold, New Jersey. Equine ferritin (MW 470,000) was purchased from Grand Island Biological Company. Human immunoglobulin M (IgM; MW 1 million) was a gift from Dr. P. H. Plotz, N I A M D D , and low density lipoprotein (LDL; MW 3.2 million) was a gift from Dr. H. R. Sloan, N H L I , NIH. Reagents. Acrylamide (Cat. No. 0019) and BIS (Cat. No. 0719) were purchased from Polysciences Inc., Warrington, Pennsylvania. D A T D 1 (Cat. No. BO-71005) was purchased from General Biochemicals, Chagrin Falls, Ohio, or (Cat. No. 161-0620) from Bio-Rad, Richmond, California. All other chemicals were reagent grade. PAGE, IFPA, and ITPPA. Stacking gels of 2.5-30% T, 15-30% C (BIS or DATD) were used in multiphasic buffer system 2950 (operative pl-i, 10.4) (8,9, Appendixl). These gels are designated as "highly crosslinked." For comparison, 4% C (BIS) gels ranging from 3 to 25% T were used in the same buffer system. Initiator mixtures consisted of 5 × 10-4% riboflavin, 0.015% potassium persulfate, 1 /zl of Temedl/ml at 0°C. The polymerization procedure and verification of stacking was carried out as described (7,8). Preparative ITPPA was performed in 5% T, 15% C (DATD) gels in preparative apparatus C and D as reported elsewhere (6,10). I F P A was performed in the same gel types as described (11). Gels containing synthetic ampholytes were stained according to the procedure of Reisner et al. (12). 2 The term "nonrestrictive,"as used in the context of this report, should not be perceived in absolute terms but relative to the propagationrate of the movingboundary(19). It is emphasized that gels are necessarily restrictive to even small molecules, such as buffer ions (20), urea, and D20 (13,2l).
34
BAUMANN
AND CHRAMBACH
RESULTS
The mechanical stability and wall adherence of highly crosslinked gels is substantially increased when BIS is substituted by D A T D as the crosslinking agent. In IFPA, gels highly crosslinked with BIS had a tendency to fall out of the glass tubes, while gels crosslinked with DATD showed no vertical displacement under identical conditions. In addition, pH-dependent shrinking and swelling of the gels did not lead to visible separation from the glass wall in DATD-crosslinked gels, but wall separation was always noted in gels highly crosslinked with BIS. The most impressive evidence for these improved mechanical properties is apparent in preparative ITPPA (6): Large 50-200-ml gel blocks could be maintained for several days without noticeable distortion of the surface or evidence of wall separation. An added advantage is the complete transparency of highly DATD-crosslinked gels in contrast to the white opacity of highly BIS-crosslinked gels. These advantages of DATD-crosslinked gels are in part offset by an increase in molecular sieving compared to gels highly crosslinked with BIS. The relative magnitude of molecular sieving effects in highly DATDcrosslinked gels, highly crosslinked-BIS gels, and conventional (2-5% crosslinked) BIS gels was evaluated by measurement of Rs in P A G E at pH 10.4, using stacking gels of varying concentrations. The results for a series of proteins with molecular weights ranging from 23,000 to 3.2 million are given in Tables 1 and 2. The retardation coefficient, Ka, is the measure of pore size in gel electrophoresis under any given set of conditions of pH, temperature, and propagation rate of the reference moving boundary (13). KR values are smaller in 15% DATD-crosslinked gels than in 4% BIS-crosslinked gels, indicating that the latter are more TABLEI RETARDATION COEFFICIENTS (KR) OF STANDARD PROTEINS IN 15% DATD-CRosSLINKED AND 4% BIS-CRosSLINKED GELS (BUFFER SYSTEM NO. 2950, PHASE ZETA, PH 10.4, 0°C) Kn Protein oPRL Ovalbumin BSA Phosphorylase B Aldolase Catalase Ferritin IgM LDL
Molecular Weight 2.3 4.4 6.7 1.0 1.5 2.4 4.7 1.0 3.2
x x x x x x x x x
104 104 104 l0 s 105 105 l0 s 106 10n
15% C ( D A T D )
4% C (BIS)
0.018 0.020 0.036 0.045 0.061 0.069 0.105 0.219 0.234
0.047 0.060 0.106 0.106 0.147 0.146 0.365 0.377 1.217
MECHANICALLY STABLE STACKING GELS
35
TABLE 2 MAXIMALGEL CONCENTRATIONSOF 15% DATD-CROSSLINKEDGELS WHICH PROVIDEAN Re = 1.00 AT PH 10.4 (LOWER STACKINGLIMIT, RM (1, ZETA)= 0.046) FOR THE PROTEINS SHOWNa Gel Concentration (% T) Protein oPRL Ovalbumin BSA Phosphorylase B Aldolase Catalase Ferritin IgM LDL
MolecularWeight 2.3 4.4 6.7 1.0 1.5 2.4 4.7 1.0 3.2
× × × x x × × × ×
104 104 10a 10~ 105 105 105 106 106
15% C (DATD)
4% C (BIS)
20 18 15 10 9 7.5 6 5 3.5
11 10 10 7.5 6 5 5 3 < 3
a An Rs = 1.00 of a protein indicates that the protein is stacked within a moving boundary migrating at a particular velocity. When this velocity is low, such as in the buffer system selected here, proteins are more likely to exhibit an Rs = 1.00. Although under these circumstances the protein has a mobility in the range of the buffer constituents, an Rs of 1.00 does not necessarily indicate "absence" of molecular sieving effects. An increase of the velocity of the moving boundary [RM (1, ZETA)] by using another buffer system would eventually lead to unstacking of the protein at the same gel concentration. Therefore, the "Re = 1.00 test" represents only a rough guide for "nonrestrictiveness." An absolute measure for molecular sieving effects can only be obtained by the more laborious procedure of determining KB.
r e s t r i c t i v e . A s a p r a c t i c a l g u i d e , t h e m a x i m a l gel c o n c e n t r a t i o n s t h a t a r e n o n r e s t r i c t i v e to a s e r i e s o f p r o t e i n s w i t h d i f f e r e n t m o l e c u l a r w e i g h t s a r e s h o w n in T a b l e 2. A c o m p a r i s o n b e t w e e n t h e p o r e s i z e s o f 15% C D A T D g e l s a n d 15% C B I S gels w a s m a d e using L D L . L D L m i g r a t e d w i t h R s = 1.00 in 15% B I S - c r o s s l i n k e d gels up to a gel c o n c e n t r a t i o n o f 9 % T . W h i l e a n i n c r e a s i n g d e g r e e o f c r o s s l i n k i n g up to 50% C ( B I S ) h a s b e e n r e p o r t e d to r e s u l t in p r o g r e s s i v e l y l a r g e r p o r e size (3), t h e p o r e size o f D A T D - c r o s s l i n k e d gels a p p e a r e d t o r e m a i n a p p r o x i m a t e l y c o n s t a n t f r o m 15% t o 30% C. Figure 1 illustrates the advantages of highly DATD-crosslinked gels in I F P A as c o m p a r e d to a c o n v e n t i o n a l 4 % T , 4% C ( B I S ) gel. I t is c l e a r l y a p p a r e n t t h a t t h e p r o t e i n s a r e m o r e s e v e r e l y r e t a r d e d in t h e i r m i g r a t i o n t o w a r d t h e i s o e l e c t r i c p o i n t in t h e c o n v e n t i o n a l 4 % B I S - c r o s s l i n k e d gel t h a n in t h e h i g h l y c r o s s l i n k e d D A T D gel. L D L d i d n o t e n t e r t h e 4 % T , 4 % C ( B I S ) g e l e x c e p t for a v e r y m i n o r c o m p o n e n t . A n a t t e m p t w a s m a d e to c o m b i n e t h e a d v a n t a g e s o f B I S gels a n d D A T D gels ( o p e n p o r e size a n d m e c h a n i c a l s t a b i l i t y , r e s p e c t i v e l y ) b y
36
BAUMANN AND CHRAMBACH
$-'?/'
A.
B.
A.
B.
Fro. 1. Protein patterns after 30 min of I F P A (ampholyte pI-range 3.5-10, 0°C) at 200 V. The two gels on the left side contain IgM, those on the right LDL. The proteins were applied at the top. The anode is placed at the bottom. (A) 4% T, 4% C (BIS) gel; (B) 5% T, 15% C (DATD) gel. Arrows indicate migration distances/30 rain for representative bands.
using mixtures of the two crosslinking agents in various proportions. It was found that the admixture of 0.5-3% D A T D to B1S (w/w) resulted in improved wall adherence without significant decrease in pore size. Opacity of these gels decreased with increasing percentage of DATD. LDL remained unrestricted in such gels up to a gel concentration of 9% T, 15% C. Higher percentages of DATD, however, resulted in gel properties much like those of pure DATD-crosslinked gels. DISCUSSION The need for a nonrestrictive gel in stacking, ITPPA, and IFPA derives from the fact that significant retardation of the compound under investigation results in unstacking in the first two cases and, in the latter, inability to reach the isoelectric position during the life-span of the pH gradient. The previous finding that the effective pore size of polyacrylamide gels could be increased significantly by increasing the degree of crosslinking seemed to provide a tool for making such nonrestrictive gels (3). However, the mechanical properties of BIS-crosslinked gels in the range of interest (10-50% C) deteriorate with increasing degree of crosslinking. Such gels become increasingly brittle and exhibit decreasing wall adherence. They are also opaque and adsorb dyes used for the staining of protein bands. Previous experience with polyacrylamide gels crosslinked with D A T D and ethylene-diacrylate for purposes of easy solubilization (14,15)
M E C H A N I C A L L Y STABLE S T A C K I N G GELS
37
prompted us to investigate the applicability of highly crosslinked D A T D gels to ITPPA and IFPA. It appears from the present data that highly DATD-crosslinked gels (e.g., a 5% T, 15% C ( D A T D ) gel) provide desirable mechanical stability, wall adherence, and a pore size sufficiently open to permit the unrestricted passage of compounds not exceeding a molecular weight of 0.5 million. Such gels are not brittle, support their own weight even in large blocks, and can be sliced with adequate precision (16). The availability of a generally applicable IFPA and ITPPA gel obviates the need to investigate, in each particular instance, the maximal gel concentration that is nonrestrictive to the compound of interest. The 5% T, 15% C (DATD) gel should replace previously used mechanically stable, but more restrictive, gels both in IFPA (17) and in ITPPA (18), which may result in unstacking or inability to reach the isoelectric position. ACKNOWLEDGMENT This work was supported in part by "Stiftung fiir Biologisch-Medizinische Stipendien," Switzerland, APPENDIX 1 MULTIPHASIC BUFFER SYSTEM No. 2950
Upper buffer
Stacking gel buffer
~/-Aminobutyric acid 2-Amino-2-methylpropane-1,3-diol HzO
4.12 g 4.34 g to 1000 ml
1 N H2SO4 H20
21.3 ml 2.30 g to 100 ml
1 N HCI 2-Amino-2-methylpropane- 1,3-diol HzO
50.0 ml 6.57 g to 1000 ml
2-Amino-2-methylpropane-1,3-diol Lower buffer
REFERENCES 1. 2. 3. 4. 5.
Ornstein, L. L. (1964)Ann. N . Y . Acad. Sci. 121, 321. Davis, B. J. (1964)Ann. N . Y . Acad. Sci. 121, 404. Rodbard, D., Levitov, C., and Chrambach, A. (1972) Separ. Sci. 7, 705. Chrambach, A., and Rodbard, D. (1971)Science 172, 440. Chrambach, A., Doerr, P., Finlayson, G. R., Miles, L. E. M., Sherins, R., and Rodbard, D. (1973)Ann. N . Y . Acad. Sci. 209, 44. 6. Baumann, G., and Chrambach, A., Proc. Nat. Acad. Sci. USA, in press. 7. Rodbard, D., and Chrambach, A. (1971) Anal. Biochem. 40, 95. 8. Rodbard, D., and Chrambach, A. (1974) in Electrophoresis and Isoelectric Focusing on Polyacrylamide Gel (Allen, R. C., and Maurer, H. R., eds.), p. 62, Walter de Gruyter, Berlin.
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BAUMANN AND CHRAMBACH
9. Jovin, T. M. (1973)Biochemist~ 12, 871. 10. Kapadia, G., and Chrambach, A. (1972)Anal. Biochem. 48, 90; Appendix III; Preparative PAGE Procedure: Apparatus C and D. 11. Baumann, G., and Chrambach, A. (1975) in Progress in Isoelectric Focusing and Isotachophoresis (Righetti, P. G., ed.), Elsevier, Excerpta Medica, North-Holland, Assoc. Sci. Publ., Amsterdam, in press. 12. Reisner, A. H., Nemes, P., and Bucholtz, C. (1975)Anal. Biochem. 64, 509. 13. Rodbard, D., and Chrambach, A. (1970) Proc. Nat. Acad. Sci. USA 65, 970. 14. Anker, H. S. (1970) F E B S Lett. 7, 293. 15. Choules, G. L., and Zimm, B. H. (1965)Anal. Biochem. 13, 336. 16. Peterson, J. I., Tipton, H. W., and Chrambach, A. (1974)Anal. Biochem. 62, 274. 17. Righetti, P. J., and Drysdale J. W. (1974) J. Chromatogr. 98, 271. 18. Svendsen, P. J., and Rose. C. (1970) Sci. Tools 17, 13. 19. Chrambach, A., and Baumann, G. (1975) in Isoelectric Focusing (Catsimpoolas, N., ed.), Academic Press, New York, in press. 20. Chrambach, A., Hearing, E., Lunney, J., and Rodbard, D., (1972)Separ. Sci. 7 725. 21. White, M. L., and Dorion, G. H. (1961) J. Polym. Sci. 55, 731.
