Journal o f l m m u n o l o g i c a l Methods, 29 (1979) 97--100
97
© Elsevier/North-Holland Biomedical Press
DIRECT TISSUE ISOELECTRIC FOCUSING IN AGAROSE
C.A. SARAVIS, M. O'BRIEN and N. ZAMCHECK Mallory Gastrointestinal Research Laboratory, Sears Surgical Laboratory and Harvard Surgical 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 are isoelectric focusing small amounts of tissues placed directly on electroendosmosis-free agarose gels instead of using the saline extracts of tissue homogenates. More numerous proteins are extracted during isoelectric focusing of the solid tissues than present in the saline extracts of the same tissues.
INTRODUCTION T h e q u a l i t a t i v e a n d q u a n t i t a t i v e analysis o f soluble m a c r o m o l e c u l a r proteins in small a m o u n t s o f tissue is n o w possible. R e c e n t l y we d e s c r i b e d a t e c h n i q u e f o r isoelectric f o c u s i n g in agarose. H e r e , we s h o w h o w it can be u s e d f o r t h e d i r e c t isoelectric f o c u s i n g o f tissue s e c t i o n s r a t h e r t h a n e x t r a c t s . P r o t e i n s m o v i n g f r o m t h e tissue s e c t i o n s d u r i n g isoelectric f o c u s i n g w e r e m o r e n u m e r o u s t h a n w e r e p r e s e n t in saline e x t r a c t s o f t h e s a m e k i n d o f tissues. W i e m e ( 1 9 5 9 ) d e s c r i b e d d i r e c t tissue e l e c t r o p h o r e s i s in agar (simple e l e c t r o p h o r e s i s ) , t h a t u s e d small a m o u n t s o f tissue, m a k i n g clinical b i o p s y m a t e r i a l available f o r analysis. This r a p i d t e c h n i q u e a v o i d e d having t o hom o g e n i z e a n d e x t r a c t tissues f o r analysis. T h e e l e c t r o p h o r e t i c e x t r a c t i o n was essentially i n s t a n t a n e o u s . T h e availability o f d i r e c t tissue isoelectric f o c u s i n g ( D T I F ) m a d e possible significantly b e t t e r s e p a r a t i o n o f soluble tissue proteins. I s o e l e c t r i c f o c u s i n g significantly i m p r o v e d t h e z o n i n g o f t h e s e p a r a t e d c o m p o n e n t s , a n d o v e r l o a d i n g o f t h e r e a c t i o n p l a t e was m i n i m i z e d . MATERIALS AND METHODS T h e p r e p a r a t i o n o f t h e agarose isoelectric f o c u s i n g plates was d e s c r i b e d in a n o t h e r c o m m u n i c a t i o n (Saravis a n d Z a m c h e c k , 1 9 7 9 ) . Q u a n t i t a t i v e i n s e r t i o n o f tissue s e c t i o n s i n t o t h e electric field allowing tissue c o m p a r i s o n s was a c c o m p l i s h e d b y placing a m a s k c o n t a i n i n g o p e n i n g s o f f i x e d size o n t h e gel s u r f a c e a n d p o s i t i o n i n g u n i f o r m l y t h i c k tissue s e c t i o n s o v e r the openings. A 2 mil t h i c k M y l a r m a s k c o n t a i n i n g 2 m m X 6 m m load-
98
Fig. 1. Isoelectric focusing of a tissue section extract of a colonic adenocarcinoma (2).
(1)
and an equivalent
amount
of a saline
99 ing slits was p l a c e d o n t h e gel surface a n d 4 - - 8 p m t h i c k tissue sections (fresh f r o z e n ) p l a c e d o v e r t h e l o a d i n g slit openings. I s o e l e c t r i c f o c u s i n g was perf o r m e d at 2 4 0 V f o r 10 m i n at r o o m t e m p e r a t u r e using a c o n s t a n t v o l t a g e s u p p l y w i t h a 1 5 , 0 0 0 ,,2 resistor at t h e a n o d e in series with t h e electrop h o r e s i s c h a m b e r . A t t h e e n d o f this t i m e , t h e m a s k w i t h tissue sections was lifted a n d r e m o v e d f r o m the gel surface, t h e n e l e c t r o p h o r e s i s c o n t i n u e d at 3 9 0 V f o r 80 m i n . A l t e r n a t i v e l y , in o t h e r runs, 10 m A w e r e a p p l i e d f o r 10 m i n , t h e m a s k and tissue s e c t i o n s r e m o v e d , a n d 25 W c o n s t a n t p o w e r a p p l i e d f o r 30 m i n at 5°C. T h e p l a t e was fixed, a m p h o l y t e s r e m o v e d , a n d t h e p l a t e stained a n d cleared (Saravis a n d Z a m c h e c k , 1979). RESULTS AND DISCUSSION G o o d s e p a r a t i o n o f t h e p r o t e i n s o f a c o l o n i c a d e n o c a r c i n o m a tissue sect i o n was o b t a i n e d using D T I F {Fig. 1).
10
20
30
40
50
60
Fig. 2. Isoelectric focusing of tissue sections of a colonic adenocarcinoma left on the reaction plate for 10, 20, 30, 40, 50 and 60 rnin.
100 A n o t h e r s e c t i o n o f t h e s a m e c o l o n i c a d e n o c a r c i n o m a was t a k e n a n d m a n ually h o m o g e n i z e d a n d e x t r a c t e d w i t h saline at 4 °C. T e n m i c r o l i t e r s o f t h e t u m o r e x t r a c t (60 m g / m l ) w e r e isoelectric f o c u s e d s i m u l t a n e o u s l y w i t h t h e tissue section. C o m p a r i s o n s h o w e d t h a t t h e direct tissue isoelectric f o c u s i n g gave a g r e a t e r n u m b e r o f d i f f e r e n t p r o t e i n s , a n d t h a t m a n y o f t h o s e in c o m m o n w i t h t h e t u m o r e x t r a c t s w e r e q u a n t i t a t i v e l y greater. I d e n t i f i c a t i o n o f these p r o t e i n s is in progress as well as c o m p a r i s o n o f n o r m a l a n d m a l i g n a n t tissues b y D T I F a n d i m m u n o f i x a t i o n o f t h e s e p a r a t e d c o n s t i t u e n t s . D T I F o f t u m o r tissue p l a c e d f o r increasing p e r i o d s o f t i m e on t h e isoelectric f o c u s i n g p l a t e t h e n r e m o v e d s h o w e d t h a t tissue a p p l i e d f o r 1 0 - - 3 0 rain gave s u f f i c i e n t p r o t e i n f o r g o o d s e p a r a t i o n p a t t e r n s (Fig. 2). D T I F is u s e d in o u r l a b o r a t o r y as a s i m p l e a n d useful t e c h n i q u e t o directly a n d r e p r o d u c i b l y e x t r a c t a n d f o c u s t h e soluble p r o t e i n s o f b i o p s y tissues, instead o f using tissue h o m o g e n a t e s . 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 , C o n t r a c t C B 6 4 0 7 1 and Grant CA18620 from the National Cancer Institute, National Institutes of Health. We t h a n k Messrs. R. C o o k , H. Witt a n d Dr. D. R e n n f o r t h e i r advice a n d Messrs. C. C u n n i n g h a m a n d P. Marasco 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 Axelsen, N.H., 1973, in: A Manual of Quantitative Immunoelectrophoresis, eds. N.H. Axelsen, J. Kroll and B. Weeke (Universitetsforlaget, Oslo). Saravis, C.A. and N. Zamcheck, 1979, J. Immunol. Methods 29, 91. Wieme, R.J., 1959, Studies on Agar Gel Electrophoresis Techniques--Applications (Arscia, Brussels).
97
© Elsevier/North-Holland Biomedical Press
DIRECT TISSUE ISOELECTRIC FOCUSING IN AGAROSE
C.A. SARAVIS, M. O'BRIEN and N. ZAMCHECK Mallory Gastrointestinal Research Laboratory, Sears Surgical Laboratory and Harvard Surgical 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 are isoelectric focusing small amounts of tissues placed directly on electroendosmosis-free agarose gels instead of using the saline extracts of tissue homogenates. More numerous proteins are extracted during isoelectric focusing of the solid tissues than present in the saline extracts of the same tissues.
INTRODUCTION T h e q u a l i t a t i v e a n d q u a n t i t a t i v e analysis o f soluble m a c r o m o l e c u l a r proteins in small a m o u n t s o f tissue is n o w possible. R e c e n t l y we d e s c r i b e d a t e c h n i q u e f o r isoelectric f o c u s i n g in agarose. H e r e , we s h o w h o w it can be u s e d f o r t h e d i r e c t isoelectric f o c u s i n g o f tissue s e c t i o n s r a t h e r t h a n e x t r a c t s . P r o t e i n s m o v i n g f r o m t h e tissue s e c t i o n s d u r i n g isoelectric f o c u s i n g w e r e m o r e n u m e r o u s t h a n w e r e p r e s e n t in saline e x t r a c t s o f t h e s a m e k i n d o f tissues. W i e m e ( 1 9 5 9 ) d e s c r i b e d d i r e c t tissue e l e c t r o p h o r e s i s in agar (simple e l e c t r o p h o r e s i s ) , t h a t u s e d small a m o u n t s o f tissue, m a k i n g clinical b i o p s y m a t e r i a l available f o r analysis. This r a p i d t e c h n i q u e a v o i d e d having t o hom o g e n i z e a n d e x t r a c t tissues f o r analysis. T h e e l e c t r o p h o r e t i c e x t r a c t i o n was essentially i n s t a n t a n e o u s . T h e availability o f d i r e c t tissue isoelectric f o c u s i n g ( D T I F ) m a d e possible significantly b e t t e r s e p a r a t i o n o f soluble tissue proteins. I s o e l e c t r i c f o c u s i n g significantly i m p r o v e d t h e z o n i n g o f t h e s e p a r a t e d c o m p o n e n t s , a n d o v e r l o a d i n g o f t h e r e a c t i o n p l a t e was m i n i m i z e d . MATERIALS AND METHODS T h e p r e p a r a t i o n o f t h e agarose isoelectric f o c u s i n g plates was d e s c r i b e d in a n o t h e r c o m m u n i c a t i o n (Saravis a n d Z a m c h e c k , 1 9 7 9 ) . Q u a n t i t a t i v e i n s e r t i o n o f tissue s e c t i o n s i n t o t h e electric field allowing tissue c o m p a r i s o n s was a c c o m p l i s h e d b y placing a m a s k c o n t a i n i n g o p e n i n g s o f f i x e d size o n t h e gel s u r f a c e a n d p o s i t i o n i n g u n i f o r m l y t h i c k tissue s e c t i o n s o v e r the openings. A 2 mil t h i c k M y l a r m a s k c o n t a i n i n g 2 m m X 6 m m load-
98
Fig. 1. Isoelectric focusing of a tissue section extract of a colonic adenocarcinoma (2).
(1)
and an equivalent
amount
of a saline
99 ing slits was p l a c e d o n t h e gel surface a n d 4 - - 8 p m t h i c k tissue sections (fresh f r o z e n ) p l a c e d o v e r t h e l o a d i n g slit openings. I s o e l e c t r i c f o c u s i n g was perf o r m e d at 2 4 0 V f o r 10 m i n at r o o m t e m p e r a t u r e using a c o n s t a n t v o l t a g e s u p p l y w i t h a 1 5 , 0 0 0 ,,2 resistor at t h e a n o d e in series with t h e electrop h o r e s i s c h a m b e r . A t t h e e n d o f this t i m e , t h e m a s k w i t h tissue sections was lifted a n d r e m o v e d f r o m the gel surface, t h e n e l e c t r o p h o r e s i s c o n t i n u e d at 3 9 0 V f o r 80 m i n . A l t e r n a t i v e l y , in o t h e r runs, 10 m A w e r e a p p l i e d f o r 10 m i n , t h e m a s k and tissue s e c t i o n s r e m o v e d , a n d 25 W c o n s t a n t p o w e r a p p l i e d f o r 30 m i n at 5°C. T h e p l a t e was fixed, a m p h o l y t e s r e m o v e d , a n d t h e p l a t e stained a n d cleared (Saravis a n d Z a m c h e c k , 1979). RESULTS AND DISCUSSION G o o d s e p a r a t i o n o f t h e p r o t e i n s o f a c o l o n i c a d e n o c a r c i n o m a tissue sect i o n was o b t a i n e d using D T I F {Fig. 1).
10
20
30
40
50
60
Fig. 2. Isoelectric focusing of tissue sections of a colonic adenocarcinoma left on the reaction plate for 10, 20, 30, 40, 50 and 60 rnin.
100 A n o t h e r s e c t i o n o f t h e s a m e c o l o n i c a d e n o c a r c i n o m a was t a k e n a n d m a n ually h o m o g e n i z e d a n d e x t r a c t e d w i t h saline at 4 °C. T e n m i c r o l i t e r s o f t h e t u m o r e x t r a c t (60 m g / m l ) w e r e isoelectric f o c u s e d s i m u l t a n e o u s l y w i t h t h e tissue section. C o m p a r i s o n s h o w e d t h a t t h e direct tissue isoelectric f o c u s i n g gave a g r e a t e r n u m b e r o f d i f f e r e n t p r o t e i n s , a n d t h a t m a n y o f t h o s e in c o m m o n w i t h t h e t u m o r e x t r a c t s w e r e q u a n t i t a t i v e l y greater. I d e n t i f i c a t i o n o f these p r o t e i n s is in progress as well as c o m p a r i s o n o f n o r m a l a n d m a l i g n a n t tissues b y D T I F a n d i m m u n o f i x a t i o n o f t h e s e p a r a t e d c o n s t i t u e n t s . D T I F o f t u m o r tissue p l a c e d f o r increasing p e r i o d s o f t i m e on t h e isoelectric f o c u s i n g p l a t e t h e n r e m o v e d s h o w e d t h a t tissue a p p l i e d f o r 1 0 - - 3 0 rain gave s u f f i c i e n t p r o t e i n f o r g o o d s e p a r a t i o n p a t t e r n s (Fig. 2). D T I F is u s e d in o u r l a b o r a t o r y as a s i m p l e a n d useful t e c h n i q u e t o directly a n d r e p r o d u c i b l y e x t r a c t a n d f o c u s t h e soluble p r o t e i n s o f b i o p s y tissues, instead o f using tissue h o m o g e n a t e s . 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 , C o n t r a c t C B 6 4 0 7 1 and Grant CA18620 from the National Cancer Institute, National Institutes of Health. We t h a n k Messrs. R. C o o k , H. Witt a n d Dr. D. R e n n f o r t h e i r advice a n d Messrs. C. C u n n i n g h a m a n d P. Marasco 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 Axelsen, N.H., 1973, in: A Manual of Quantitative Immunoelectrophoresis, eds. N.H. Axelsen, J. Kroll and B. Weeke (Universitetsforlaget, Oslo). Saravis, C.A. and N. Zamcheck, 1979, J. Immunol. Methods 29, 91. Wieme, R.J., 1959, Studies on Agar Gel Electrophoresis Techniques--Applications (Arscia, Brussels).
