Journal o f Immunological Methods, 29 (1979) 91--96
91
(2) Elsevier/North-ttolland Biomedical Press
I S O E L E C T R I C F O C U S I N G IN A G A R O S E
C.A SARAVIS and N. ZAMCHECK The Mallory Gastrointestinal Research Laboratory, Sears Surgical Laboratory and Har,,atd Su,:gical Unit, Boston City Hospital, Boston, MA, Departments o f Surgery and Medicine, Harvard Medical School, and Department o f Pathology, Boston University School o f Medicine, Boston, MA 02118, U.S.A.
(Received 2 January 1979, accepted 22 March 1979)
We describe here the use of a new agarose for isoelectric focusing of body fluids and tissue proteins. Macromolecular proteins even when significantly greater in size than 1 x 10 5 m~)l.wt, were easily and rapidly focused.
INTRODUCTION T h e use o f agarose gels with n o d e m o n s t r a b l e e l e c t r o e n d o s m o s i s in t h e analytical isoelectric focusing t e c h n i q u e d e s c r i b e d in this p a p e r p e r m i t t e d s e p a r a t i o n o f m a c r o m o l e c u l a r p r o t e i n s o f n o r m a l h u m a n plasma and tissue, as well as m a l i g n a n t h u m a n ascites and t u m o r s n o t possible with p o l y a c r y l amide gels. T h e agarose used was p r o v i d e d b y Marine Colloids Division o f FMC C o r p o r a t i o n , R o c k l a n d , ME, and is c o m m e r c i a l l y available f r o m t h e m as Isogel. Previously available agarose e x h i b i t e d high e l e c t r o e n d o s m o s i s (Quast, 1971) and did n o t give stable p H gradients and p a t t e r n s in isoelectric focusing (Riley and C o l e m a n , 1 9 6 8 ; Catsimpoolas, 1 9 6 9 ) . Chemical m o d i f ication o f agarose to r e d u c e e l e c t r o e n d o s m o s i s (L££s, 1 9 7 2 ; J o h a n s s o n and Hjert6n, 1974) was r e p o r t e d to be effective b u t the agarose has n o t been c o m m e r c i a l l y available to the m a n y o t h e r investigators w h o would w a n t to use it. T h e r e f o r e , Isogel was d e v e l o p e d for readily p e r f o r m e d isoelectric focusing o f proteins with m o l e c u l a r weights over 5 0 0 , 0 0 0 . T h e advantages o f using Isogel for isoelectric focusing in investigations o f t u m o r markers of h u m a n c a n c e r is described in this c o m m u n i c a t i o n . MATERIALS AND METHODS The agarose and G e l b o n d , t h e h y d r o p h i l i c p o l y e s t e r s u p p o r t , were f r o m Marine Colloids. Carrier a m p h o l y t e s were p u r c h a s e d f r o m L K B P r o d u k t u r , Pharmacia, or Bio-Rad L a b o r a t o r i e s . All o t h e r reagents were the highest grade available. Isoelectric focusing was p e r f o r m e d in e q u i p m e n t p r o d u c e d in this labora-
92 tory by C.A. Saravis. However, most available commercial isoelectric focusing equipment could have been used. Fifteen ml agarose aliquots, prepared from 0.5% agarose in deionized water and stored at 4 ° C, were brought into solution in a boiling water bath, put at 56 ° C, mixed with ampholytes to 2.5%, and gelled on a 10 cm × 10 cm piece of clear, flexible, 7 mil thick, polyester-based plastic film (Gelbond) specially treated on one side for adherence of gels. The formed plates were 'aged' at 4°C for 1 h before use to obtain reproducible electrophoretic characteristics. Synthetic cellulose sponge wicks (duPont de Nemours EI and Co.) wet with anode and cathode solutions (e.g. acid, base, amino acids, ampholytes, etc.) were placed on the gel surface at the ends of the plate, and platinum electrode wires placed on top of the wicks. Alternatively, when using an ampholyte range of pH 2.5--10, the electrodes were applied directly to the surface of the gel, and I N NaOH placed in a container within the electrophoresis chamber to absorb CO2 resulting in pattern improvement at the basic end of the gel plate. A 2 mil thick Mylar sheet containing loading slits was placed on the gel surface and 1 pl samples placed in each opening. The samples were focused at 240 V for 10 min at room temperature using a constant voltage supply with a 15,000 ~ resistor at the anode in series with the electrophoresis chamber (Awdeh, 1972). The mask was removed, and the samples further separated at 390 V for 80 min. Alternatively significantly faster focusing was obtained by initially applying 10 mA for 10 min, removing the mask and focusing the samples at 25 W constant power (1000 V limiting) for 30 min at 5°C. The plates were immediately placed in fixative solution (containing methanol 30%, trichloroacetic acid 5%, and sulphosalicylic acid 3.5%) for 10 min. Filter paper No. 577 (Schleicher and Schuell, Keene, NH) pre-wet with water was placed on each gel surface, several (e.g. Catsimpoolas, 1969) layers of absorbent paper toweling placed on top of the filter paper, and ampholytes and water removed from the gel (approximately 10--15 min). The plates were dried with warm air, the filter paper wet with deionized water and removed, and the agarose plates thoroughly dried with warm air at 60 °C. The dried plates were stained with Coomassie Blue R-250, 0.5%, 60°C, for 10--15 min, rinsed with 8% acetic acid containing 25% methanol for 1--2 min, evaluated, and attached to the reports without further treatment. RESULTS AND DISCUSSION AIF gave good separation of (a) the proteins of a 5 pm thick tissue section of a colonic adenocarcinoma applied directly to the surface of the agarose plate (Saravis et al., 1979), (b) the ascitic fluid from a patient with a colonic adenocarcinoma, and (c) normal h u m a n plasma (Fig. 1). Similar separation patterns were obtained when 3 malignant h u m a n ascites (1 colonic adenocar-
93 m
W
1
-
2"
Fig. 1. A tissue s e c t i o n o f a h u m a n c o l o n i c a d e n o c a r c i n o m a placed o n the gel surface: 1, ascites fluid (1 pl) f r o m a p a t i e n t w i t h c o l o n a d e n o c a r c i n o m a ; 2, n o r m a l h u m a n plasma (1.5 pl); 3, c o n c e n t r a t i o n s were c h o s e n to e n h a n c e the d e m o n s t r a t i o n o f b a n d s at the basic e n d o f the r e a c t i o n plate.
cinoma, 1 lung cancer, 1 l y m p h o m a ) were placed on the same plate either cathodically and anodically, and isoelectric focused (Fig. 2). A linear pH gradient between the electrodes was obtained and could be used to measure the pH o f the separated proteins. F u r t h e r characterization and identification of these proteins are in progress. The t u m o r marker, zinc glycinate marker (ZGM) (Pusztaszeri et al., 1976) with a pH of 5.7 (Safaris and Zamcheck,
94
lO 9: 8 ~ii i~iii~iii!!i{~i~ ¸
7
6' "I"
O.
5 4 3 1 t
,
2
3
,
.
~
.
~
c .M
6 .
7 .
8
9
0
t~
: ~i !ill i~ii~ii!ii !i!
Fig. 2. Isoelectric focusing of m a l i g n a n t ascites applied at t w o d i f f e r e n t positions. T h e p i t g r a d i e n t was d e t e r m i n e d b y removal of serial s e c t i o n s of t h e A I F plate at c o m p l e t i o n of isoelectric focusing a n d b e f o r e it was fixed a n d s t a i n e d , h o m o g e n i z i n g each s e c t i o n in I ml o f d e a e r a t e d distilled water, and m e a s u r i n g t h e pH 60 rain later. Ascitic fluids f r o m p a t i e n t s w i t h l y m p h o m a : 1, lung cancer; 2, c o l o n a d e n o c a r c i n o m a ; 3, applied (1 pl) at t h e level of t h e arrows. Fig. 3. Isoeleetric focusing of purified Z G M ( ~ 5 0 0 Hg) applied at t h e level of t h e arrow.
95 1978) and a molecular weight/>2 × 106, was used to study the efficacy of AIF in analyzing large size molecules. ZGM was used that was prepared by preparative Pevikon electrophoresis, molecular sieving on Sepharose 4B, and Con A-Sepharose, and considered purified to homogeneity by immunoelectrophoretic and physico-chemical analysis. AIF showed migration of this glycoprotein from a cathodal application point to an anodal position, and resolution into multiple lines in close approximation to each other (Fig. 3). We are studying the multiple lines, whether these result from the denaturation and fragmentation of this large, hygroscopic molecule at its isoelectric point, or whether the lines indicate association of other proteins with ZGM. LKB 3.5--10 ampholytes when supplemented with LKB 2.5--4, and LKB 5--8 ampholytes at a ratio of 3 : 1 : 1, were used in producing the patterns demonstrated. Pharmalytes 3--10 (unsupplemented) and Bio-Rad ampholytes gave good focusing of proteins using this technique.
Some advantages o f agarose over polyacrylamide for isoelectric focusing The agarose gels formed from a single, stable and non-toxic polymer are brought into solution within minutes, mixed with ampholytes and gelled. The gels do not exclude macromolecules as large as ZGM. Ampholytes are removed from reacted agarose gel plates within minutes, and the agarose gels easily dried, stained and cleared. The dried and stained separation patterns then can be attached w i t h o u t further treatment directly to the experimental record, providing ready access for analysis. In contrast, polyacrylamide which is used in most current isoelectric focusing gel techniques does not permit separation of m a n y macromolecules including ZGM which could not penetrate the gel. In addition, molecular sieving is frequently seen during isoelectric focusing and is especially undesirable since separation by pH exclusively is required. Furthermore, several reagents are needed to form the polyacrylamide gel, some of which are unstable. The polyacrylamide gels only are used 12 or more hours after polymerization. Before this time some afterpolymerization may occur. In addition, polyacrylamide gels may be significantly affected by 'aging', especially if a slow polymerizing catalyst like riboflavin is used. Furthermore, acrylamide is known to be toxic to laboratory workers (Amer. Cyanamid Company; Eastman Dataservice Catalog JJ11; LKB Application Laboratory). AIF has been subsequently followed by: (a) electrophoretic transportation of the unfixed focused molecules at right angles into antibody-containing agarose at 25 V overnight at room temperature, or at 10 V/cm for 6--8 h at 12°C, using 0.05 M barbital buffer, pH 8.2, in the electrode reservoirs, or (b) by i m m u n o f i x a t i o n of the AIF separated proteins. The availability of Isogel makes possible a wide variety of techniques and assays that previously could not be performed.
96 ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d in p a r t b y G r a n t C A 0 4 4 8 6 a n d C o n t r a c t CB64071 from The National Cancer Institute, National Institutes of Health. We t h a n k Messrs. R. C o o k , H. W i t t a n d Dr. D. R e n n , f o r t h e i r a d v i c e a n d Messrs. C. C u n n i n g h a m a n d P. M a r a s c o a n d Mrs. B. B u r k e f o r t h e i r t e c h n i c a l assistance. REFERENCES American Cyanamid Company, Process Chemicals Dept., Wayne, NJ. Awdeh, Z.L., 1972, Sci. Tools 19, 27. Awdeh, Z.L., A.R. Williamson and B.A. Askonas, 1968, Nature 219, 66. Catsimpoolas, N., 1969, Sci. Tools 16, 1. Eastman Dataservice Catalog JJ-11, Eastman Kodak Company, Rochester, NY. Johansson, B.G. and S. Hjert6n, 1974, Anal. Biochem. 59, 200. L~s, T., 1972, J. Chromatogr. 66, 347. LKB Application Laboratory, LKB Produktor AB, Stockholm, Sweden. Pusztaszeri, G., C.A. Saravis and N. Zamcheck, 1976, J. Natl. Cancer Inst. 56, 275. Quast, R., 1971, J. Chromatogr. 54,405. Riley, R.F. and M.K. Coleman, 1968, J. Lab. Clin. Med. 72, 714. Saravis, C.A. and N. Zamcheck, 1978, Cancer 42, 225. Saravis, C.A., M. O'Brien and N. Zamcheck, 1979, J. Immunol. M~thods 29, 97.
91
(2) Elsevier/North-ttolland Biomedical Press
I S O E L E C T R I C F O C U S I N G IN A G A R O S E
C.A SARAVIS and N. ZAMCHECK The Mallory Gastrointestinal Research Laboratory, Sears Surgical Laboratory and Har,,atd Su,:gical Unit, Boston City Hospital, Boston, MA, Departments o f Surgery and Medicine, Harvard Medical School, and Department o f Pathology, Boston University School o f Medicine, Boston, MA 02118, U.S.A.
(Received 2 January 1979, accepted 22 March 1979)
We describe here the use of a new agarose for isoelectric focusing of body fluids and tissue proteins. Macromolecular proteins even when significantly greater in size than 1 x 10 5 m~)l.wt, were easily and rapidly focused.
INTRODUCTION T h e use o f agarose gels with n o d e m o n s t r a b l e e l e c t r o e n d o s m o s i s in t h e analytical isoelectric focusing t e c h n i q u e d e s c r i b e d in this p a p e r p e r m i t t e d s e p a r a t i o n o f m a c r o m o l e c u l a r p r o t e i n s o f n o r m a l h u m a n plasma and tissue, as well as m a l i g n a n t h u m a n ascites and t u m o r s n o t possible with p o l y a c r y l amide gels. T h e agarose used was p r o v i d e d b y Marine Colloids Division o f FMC C o r p o r a t i o n , R o c k l a n d , ME, and is c o m m e r c i a l l y available f r o m t h e m as Isogel. Previously available agarose e x h i b i t e d high e l e c t r o e n d o s m o s i s (Quast, 1971) and did n o t give stable p H gradients and p a t t e r n s in isoelectric focusing (Riley and C o l e m a n , 1 9 6 8 ; Catsimpoolas, 1 9 6 9 ) . Chemical m o d i f ication o f agarose to r e d u c e e l e c t r o e n d o s m o s i s (L££s, 1 9 7 2 ; J o h a n s s o n and Hjert6n, 1974) was r e p o r t e d to be effective b u t the agarose has n o t been c o m m e r c i a l l y available to the m a n y o t h e r investigators w h o would w a n t to use it. T h e r e f o r e , Isogel was d e v e l o p e d for readily p e r f o r m e d isoelectric focusing o f proteins with m o l e c u l a r weights over 5 0 0 , 0 0 0 . T h e advantages o f using Isogel for isoelectric focusing in investigations o f t u m o r markers of h u m a n c a n c e r is described in this c o m m u n i c a t i o n . MATERIALS AND METHODS The agarose and G e l b o n d , t h e h y d r o p h i l i c p o l y e s t e r s u p p o r t , were f r o m Marine Colloids. Carrier a m p h o l y t e s were p u r c h a s e d f r o m L K B P r o d u k t u r , Pharmacia, or Bio-Rad L a b o r a t o r i e s . All o t h e r reagents were the highest grade available. Isoelectric focusing was p e r f o r m e d in e q u i p m e n t p r o d u c e d in this labora-
92 tory by C.A. Saravis. However, most available commercial isoelectric focusing equipment could have been used. Fifteen ml agarose aliquots, prepared from 0.5% agarose in deionized water and stored at 4 ° C, were brought into solution in a boiling water bath, put at 56 ° C, mixed with ampholytes to 2.5%, and gelled on a 10 cm × 10 cm piece of clear, flexible, 7 mil thick, polyester-based plastic film (Gelbond) specially treated on one side for adherence of gels. The formed plates were 'aged' at 4°C for 1 h before use to obtain reproducible electrophoretic characteristics. Synthetic cellulose sponge wicks (duPont de Nemours EI and Co.) wet with anode and cathode solutions (e.g. acid, base, amino acids, ampholytes, etc.) were placed on the gel surface at the ends of the plate, and platinum electrode wires placed on top of the wicks. Alternatively, when using an ampholyte range of pH 2.5--10, the electrodes were applied directly to the surface of the gel, and I N NaOH placed in a container within the electrophoresis chamber to absorb CO2 resulting in pattern improvement at the basic end of the gel plate. A 2 mil thick Mylar sheet containing loading slits was placed on the gel surface and 1 pl samples placed in each opening. The samples were focused at 240 V for 10 min at room temperature using a constant voltage supply with a 15,000 ~ resistor at the anode in series with the electrophoresis chamber (Awdeh, 1972). The mask was removed, and the samples further separated at 390 V for 80 min. Alternatively significantly faster focusing was obtained by initially applying 10 mA for 10 min, removing the mask and focusing the samples at 25 W constant power (1000 V limiting) for 30 min at 5°C. The plates were immediately placed in fixative solution (containing methanol 30%, trichloroacetic acid 5%, and sulphosalicylic acid 3.5%) for 10 min. Filter paper No. 577 (Schleicher and Schuell, Keene, NH) pre-wet with water was placed on each gel surface, several (e.g. Catsimpoolas, 1969) layers of absorbent paper toweling placed on top of the filter paper, and ampholytes and water removed from the gel (approximately 10--15 min). The plates were dried with warm air, the filter paper wet with deionized water and removed, and the agarose plates thoroughly dried with warm air at 60 °C. The dried plates were stained with Coomassie Blue R-250, 0.5%, 60°C, for 10--15 min, rinsed with 8% acetic acid containing 25% methanol for 1--2 min, evaluated, and attached to the reports without further treatment. RESULTS AND DISCUSSION AIF gave good separation of (a) the proteins of a 5 pm thick tissue section of a colonic adenocarcinoma applied directly to the surface of the agarose plate (Saravis et al., 1979), (b) the ascitic fluid from a patient with a colonic adenocarcinoma, and (c) normal h u m a n plasma (Fig. 1). Similar separation patterns were obtained when 3 malignant h u m a n ascites (1 colonic adenocar-
93 m
W
1
-
2"
Fig. 1. A tissue s e c t i o n o f a h u m a n c o l o n i c a d e n o c a r c i n o m a placed o n the gel surface: 1, ascites fluid (1 pl) f r o m a p a t i e n t w i t h c o l o n a d e n o c a r c i n o m a ; 2, n o r m a l h u m a n plasma (1.5 pl); 3, c o n c e n t r a t i o n s were c h o s e n to e n h a n c e the d e m o n s t r a t i o n o f b a n d s at the basic e n d o f the r e a c t i o n plate.
cinoma, 1 lung cancer, 1 l y m p h o m a ) were placed on the same plate either cathodically and anodically, and isoelectric focused (Fig. 2). A linear pH gradient between the electrodes was obtained and could be used to measure the pH o f the separated proteins. F u r t h e r characterization and identification of these proteins are in progress. The t u m o r marker, zinc glycinate marker (ZGM) (Pusztaszeri et al., 1976) with a pH of 5.7 (Safaris and Zamcheck,
94
lO 9: 8 ~ii i~iii~iii!!i{~i~ ¸
7
6' "I"
O.
5 4 3 1 t
,
2
3
,
.
~
.
~
c .M
6 .
7 .
8
9
0
t~
: ~i !ill i~ii~ii!ii !i!
Fig. 2. Isoelectric focusing of m a l i g n a n t ascites applied at t w o d i f f e r e n t positions. T h e p i t g r a d i e n t was d e t e r m i n e d b y removal of serial s e c t i o n s of t h e A I F plate at c o m p l e t i o n of isoelectric focusing a n d b e f o r e it was fixed a n d s t a i n e d , h o m o g e n i z i n g each s e c t i o n in I ml o f d e a e r a t e d distilled water, and m e a s u r i n g t h e pH 60 rain later. Ascitic fluids f r o m p a t i e n t s w i t h l y m p h o m a : 1, lung cancer; 2, c o l o n a d e n o c a r c i n o m a ; 3, applied (1 pl) at t h e level of t h e arrows. Fig. 3. Isoeleetric focusing of purified Z G M ( ~ 5 0 0 Hg) applied at t h e level of t h e arrow.
95 1978) and a molecular weight/>2 × 106, was used to study the efficacy of AIF in analyzing large size molecules. ZGM was used that was prepared by preparative Pevikon electrophoresis, molecular sieving on Sepharose 4B, and Con A-Sepharose, and considered purified to homogeneity by immunoelectrophoretic and physico-chemical analysis. AIF showed migration of this glycoprotein from a cathodal application point to an anodal position, and resolution into multiple lines in close approximation to each other (Fig. 3). We are studying the multiple lines, whether these result from the denaturation and fragmentation of this large, hygroscopic molecule at its isoelectric point, or whether the lines indicate association of other proteins with ZGM. LKB 3.5--10 ampholytes when supplemented with LKB 2.5--4, and LKB 5--8 ampholytes at a ratio of 3 : 1 : 1, were used in producing the patterns demonstrated. Pharmalytes 3--10 (unsupplemented) and Bio-Rad ampholytes gave good focusing of proteins using this technique.
Some advantages o f agarose over polyacrylamide for isoelectric focusing The agarose gels formed from a single, stable and non-toxic polymer are brought into solution within minutes, mixed with ampholytes and gelled. The gels do not exclude macromolecules as large as ZGM. Ampholytes are removed from reacted agarose gel plates within minutes, and the agarose gels easily dried, stained and cleared. The dried and stained separation patterns then can be attached w i t h o u t further treatment directly to the experimental record, providing ready access for analysis. In contrast, polyacrylamide which is used in most current isoelectric focusing gel techniques does not permit separation of m a n y macromolecules including ZGM which could not penetrate the gel. In addition, molecular sieving is frequently seen during isoelectric focusing and is especially undesirable since separation by pH exclusively is required. Furthermore, several reagents are needed to form the polyacrylamide gel, some of which are unstable. The polyacrylamide gels only are used 12 or more hours after polymerization. Before this time some afterpolymerization may occur. In addition, polyacrylamide gels may be significantly affected by 'aging', especially if a slow polymerizing catalyst like riboflavin is used. Furthermore, acrylamide is known to be toxic to laboratory workers (Amer. Cyanamid Company; Eastman Dataservice Catalog JJ11; LKB Application Laboratory). AIF has been subsequently followed by: (a) electrophoretic transportation of the unfixed focused molecules at right angles into antibody-containing agarose at 25 V overnight at room temperature, or at 10 V/cm for 6--8 h at 12°C, using 0.05 M barbital buffer, pH 8.2, in the electrode reservoirs, or (b) by i m m u n o f i x a t i o n of the AIF separated proteins. The availability of Isogel makes possible a wide variety of techniques and assays that previously could not be performed.
96 ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d in p a r t b y G r a n t C A 0 4 4 8 6 a n d C o n t r a c t CB64071 from The National Cancer Institute, National Institutes of Health. We t h a n k Messrs. R. C o o k , H. W i t t a n d Dr. D. R e n n , f o r t h e i r a d v i c e a n d Messrs. C. C u n n i n g h a m a n d P. M a r a s c o a n d Mrs. B. B u r k e f o r t h e i r t e c h n i c a l assistance. REFERENCES American Cyanamid Company, Process Chemicals Dept., Wayne, NJ. Awdeh, Z.L., 1972, Sci. Tools 19, 27. Awdeh, Z.L., A.R. Williamson and B.A. Askonas, 1968, Nature 219, 66. Catsimpoolas, N., 1969, Sci. Tools 16, 1. Eastman Dataservice Catalog JJ-11, Eastman Kodak Company, Rochester, NY. Johansson, B.G. and S. Hjert6n, 1974, Anal. Biochem. 59, 200. L~s, T., 1972, J. Chromatogr. 66, 347. LKB Application Laboratory, LKB Produktor AB, Stockholm, Sweden. Pusztaszeri, G., C.A. Saravis and N. Zamcheck, 1976, J. Natl. Cancer Inst. 56, 275. Quast, R., 1971, J. Chromatogr. 54,405. Riley, R.F. and M.K. Coleman, 1968, J. Lab. Clin. Med. 72, 714. Saravis, C.A. and N. Zamcheck, 1978, Cancer 42, 225. Saravis, C.A., M. O'Brien and N. Zamcheck, 1979, J. Immunol. M~thods 29, 97.
