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Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
A Rapid and Efficient Method for the Isolation of Proteins from Polyacrylamide Gels a
J. A. Braatzw & K. R. McLntire
a
a
Laboratory of Immunodiagnosis , National Cancer Institute, National Institutes of Health , Bethesda, Maryland, 20014 Published online: 05 Mar 2007.
To cite this article: J. A. Braatzw & K. R. McLntire (1977) A Rapid and Efficient Method for the Isolation of Proteins from Polyacrylamide Gels, Preparative Biochemistry, 7:6, 495-509, DOI: 10.1080/00327487708065516 To link to this article: http://dx.doi.org/10.1080/00327487708065516
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PREPARATIVE BIOCHEMISTRY, 7 ( 6 ) , 495-509
(1977)
A RAPID AND EFFICIENT METHOD FOR THE ISOLATION OF PROTEINS FROM POLYACRYLAMIDE GELS
Downloaded by [McGill University Library] at 22:10 02 February 2015
J. A. Braat z and K. R. M c I n t i r e L a b o r a t o r y of Immunodiagnosis, N a t i o n a l Cancer I n s t i t u t e , N a t i o n a l I n s t i t u t e s of H e a l t h , Bet hesda, Maryland 20014 ABSTRACT A p r o c e d u r e i s d e s c r i b e d f o r t h e r a p i d and e f f i c i e n t e l e c t r o p h o r e t i c
e l u t i o n o f p r o t e i n from pol yacryl ami de g e l s which i s t h e n c o l l e c t e d i n a d i a l y s i s bag t i e d t o t h e end of a t u b e c o n t a i n i n g t h e g e l s l i c e s .
To
i l l u s t r a t e t h e method a het erogeneous p r e p a r a t i o n of a l k a l i n e phos pha ta s e was used from which a s i n g l e homogeneous component was i s o l a t e d i n s i x hours w i t h a r e c o v e r y of 86%.
The e l u t e d p r o t e i n i s c o l l e c t e d i n a volume which
can e a s i l y be k e p t below 1.5 m l , t h u s e l i m i n a t i n g t h e need f o r s ubs e que nt concentration.
The method h a s a l s o been used s u c c e s s f u l l y i n two o t h e r
systems i n which a human lung t umor-associ at ed a n t i g e n and glyc oge n synt h e t a s e from y e a s t were i s o l a t e d .
S i n c e t h e method u t i l i z e s a s t a n d a r d
a n a l y t i c a l g e l e l e c t r o p h o r e s i s a p p a r a t u s w i t h no m o d i f i c a t i o n s or a c c e s s o r i e s , i t sh o u ld be immediately a p p l i c a b l e f o r t h e i s o l a t i o n of many d i f f e r e n t p r o t e i n s from pol yacryl ami de g e l s . INTRODUCTION A n a l y t i c a l p o lyacryl ami de g e l e l e c t r o p h o r e s i s is one of t h e most powerful methods a v a i l a b l e f o r d e t e r m i n i n g t h e comple xity or p u r i t y of p r o t e i n Solutions.'
F r e q u e n t l y , however, i t becomes n e c e s s a r y t o go beyond
t h e a n a l y t i c a l s t a g e by a t t e m p t i n g t o i s o l a t e s p e c i f i c p r o t e i n bands from the gel.
For example, t h e d e m o n s t r a t i o n of enzymatic o r a n t i g e n i c a c t i v i t y
a s s o c i a t e d w i t h a p a r t i c u l a r band may r e q u i r e t h e e l u t i o n and r e c o v e r y of
495 Copyright 8 1977 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any !nformatlon storageand retrieval system. without permission in writing from the publisher.
B W T Z AND MC INTIRE
49 6 that component from the gel.
Whether for this purpose or for the prepara-
tive isolation of protein from acrylamide gels, the most commonly used procedures have been a) homogenization of the gel region containing the protein followed by extraction with buffer or b) the use of commercially available preparative gel electrophoresis
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Various preparative gel systems are available which are useful for protein purification from relatively large amounts of starting material. If the amount of protein is small, however, it may be lost i n these preparative-scale gel systems due to non-specific adsorption.
On the other
hand, extraction of protein from homogenized gel slices often results in poor recoveries and usually requires long periods for diffusion, during which inactivation or degradation can occur. A number of reports have appeared which describe the electrophoretic elution of protein from polyacrylamide fied gel electrophoresis equipment.
gel^^-^
but these all require modi-
This paper describes a simple procedure
for the electrophoretic elution of proteins from gels which makes use of standard analytical gel electrophoresis equipment. MATERIALS AND METHODS Materials
-
Acrylamide, 99+%, electrophoresis grade and N,N'-Methylene-
bisacrylamide, 99+%, electrophoresis grade were purchased from the Aldrich Chemical Co. Ammonium persulfate, Ultra Pure, was obtained from Polysciences, Inc., and N,N,N',N'-Tetramethylethylenediamine
from Eastman
Kodak Co. Coomassie Brilliant Blue 6 2 5 0 was purchased from EC Apparatus Corp. E. coli alkaline phosphatase Type 111 was obtained from Sigma. Glycogen synthetase was partially purified from yeast as previously described.'
The electrophoresis chamber was obtained from Buchler Instruments
and the power supply was a Beckman Spinco Duostat.
6 nrm i.d.
x 8 mm 0.d.
7 m m 0.d.
x 125 mm from BioRad.
Gel tubes were
x 7 5 mm from Scientific Products, and 5.5 m m i.d.
Preparation of gels
-
x
Standard pH 8.9, 7% acrylamide gels were pre-
pared according to Davis9 using ammonium persulfate to initiate p o l p e r i -
49 7
PROTEINS FROM POLYACRYLAMIDE GELS zation.
The g e l s were r o u t i n e l y c a s t t o a h e i g h t of 60 or 110 mm i n t h e 75
or 125 mm t u b e s , r e s p e c t i v e l y , a t l e a s t one day p r i o r t o u s e i n o r d e r t o a l l o w S t a c k i n g g e l s were omitte d.
t r a c e s of p e r s u l f a t e t o decompose. Electrophoresis
- A maximum volume
of 150 ~1 of sample was mixed w i t h
50 ~1 of g l y c e r o l and 3 ~1of 0.05% Bromphenol Blue and a p p l i e d t o t h e g e l .
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E l e c t r o p h o r e s i s was performed a t 4°C a t a c u r r e n t of 2 ma /tube u n t i l t h e
I n some e x p e r i -
t r a c k i n g dye was about 1 cm from t h e bottom of t h e g e l .
ments i n o r d e r t o o b t a i n b e t t e r band s e p a r s t i o n , e l e c t r o p h o r e s i s was performed on g e l s 110 mm i n l e n g t h and c o n t i n u e d for one hour a f t e r t h e t r a c k i n g dye was r un o f f t h e bottom.
Gels were removed by rimming w i t h a
2 i n c h , 20 gauge n e e d l e while i n j e c t i n g water and t h e p o s i t i o n of t h e In order t o localize
t r a c k i n g dye was marked by i n s e r t i n g a p i e c e of w ire .
t h e p r o t e i n bands, a s e p a r a t e g e l was f i x e d i n 12.5% TCA"
f o r 30 min and
s t a i n e d i n Coomassie B r i l l i a n t Blue (1:20 d i l u t i o n of a 1%aqueous s o l u t i o n wi th 12.5% TCA) f o r 30 min.
P r o t e i n bands c o n t a i n i n g a s l i t t l e as
1 0 ug of p r o t e i n c oul d be v i s u a l i z e d by t h i s r a p i d s t a i n i n g proc e dure which r e q u i r e d no d e s t a i n i n g . k e p t a t -3O'C
The g e l s c o n t a i n i n g t h e p r o t e i n t o be e l u t e d were
and t h e n a l i g n e d w i t h t h e s t a i n e d g e l .
The a p p r o p r i a t e g e l
s e c t i o n s were c u t o u t w i t h a c l e a n r a z o r and t h e p r o t e i n e l u t e d as desc r i b e d below.
Ge l s c o n t a i n i n g r a d i o l a b e l e d p r o t e i n were f r o z e n f o r one
h o u r a t -3O"C,
t h e n s l i c e d i n t o 2 mm s e c t i o n s and c ounte d i n a Beckman
BioGamma c o u n t e r .
The g e l s l i c e s c o n t a i n i n g t h e r a d i o a c t i v e t r a c e of
i n t e r e s t were t h e n s e l e c t e d and t h e p r o t e i n e l u t e d as d e s c r i b e d below. E l e c t r o p h o r e t i c e l u t i o n of p r o t e i n from g e l s l i c e s
- Acrylamide
gels
were c a s t i n t o 75 mm g l a s s t u b e s t o a h e i g h t of 40 mm, i n t h e re ma inde r o f t h e t u b e were p laced t h e g e l s l i c e s c o n t a i n i n g t h e p r o t e i n t o b e e l u t e d . The t u b e s were f i l l e d w i t h e l e c t r o p h o r e s i s b u f f e r (0.025 M T r i s , 0.2 M g l y c i n e , pH 8.3, wi t h or w i t h o u t 30% g l y c e r o l ) and t h e s l i c e s were s t a c k e d on t o p of t h e s u p p o r t g e l , c a r e bei ng t a k e n t o e l i m i n a t e a l l a i r bubble s . D i a l y s i s t u b i n g (Union C arbi de Corp.),
0.5 i n . wide, was a p p l i e d t o t h e
BRAATZ AND MC INTIRE
498 t o p of t h e tube and f i x e d i n p l a c e w i t h a 5 mm i .d.
rubbe r O-ring.
Elec-
t r o p h o r e s i s b u f f e r was added c a r e f u l l y t o avoi d a i r bubble s u n t i l t h e volume o f b u f f e r i n s i d e t h e d i a l y s i s bag p l u s t h a t c o v e r i n g t h e g e l s l i c e s was ab o u t 1 m l .
The t o p of t h e bag was t i e d s e c u r e l y w i t h s t r i n g (Fig. 1A).
The g l a s s t u b e was i n s e r t e d i n t o t h e e l e c t r o p h o r e s i s chamber which was t h e n f i l l e d with buffer.
The f i n a l arrangement is shown i n Fig. 1 B .
It was
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n e c e s s a r y a t t h i s s t a g e t o r e v e r s e t h e e l e c t r o d e s so t h a t t h e p r o t e i n s m igr a t e d toward t h e upper (anode) chamber.
Reversed e l e c t r o p h o r e s i s was
t h e n c a r r i e d o u t a t 4OC a t 2 ms/ gel f o r v a r y i n g t i m e p e r i o d s a f t e r which t h e t u b e s were removed and i n v e r t e d , and t h e d i a l y s i s bag was t h e n c a r e f u l l y removed.
The b u f f e r remaining i n t h e g l a s s column w a s added t o t h e c o n t e n t s
o f t h e d i a l y s i s bag which c a n be t i e d o f f a t t h e o p p o s i t e end and d i a l y z e d i f desired.
I n some experi ment s i t was found c o n v e n i e n t , b u t not n e c e s s a r y ,
t o run t h e f i r s t ( r e s o l v i n g ) e l e c t r o p h o r e s i s i n 5.5 mm diam. g e l s and t h e r e v e r s e d e l e c t r o p h o r e s i s i n g e l t u b e s of 6 mm i.d.
i n order t o f a c i l i t a t e
s t a c k i n g t h e g e l s l i c e s on t op of t h e s u p p o r t g e l . D e t e c t i o n of enzyme a c t i v i t y on g e l s
- Alkaline
phos pha ta s e a c t i v i t y
was d e t e c t e d on g e l s usi ng a m o d i f i c a t i o n of t h e proc e dure of A lle n and Hyncik."
A f t e r e l e c t r o p h o r e s i s g e l s were immersed i n 10 m l of 0.5 M T r i s ,
pH 9.0, c o n t a i n i n g 1 mM p-ni t rophenyl phosphat e and 0.2 M CaCl a t room te mp e r a tu r e .
2
f o r 10 min
The g e l s were t h e n r i n s e d w i t h d i s t i l l e d water and
immersed i n 10 m l of 0.1 M T ri s-mal eat e,
pH 7.0,
c o n t a i n i n g 3 d Pb(N03)2
f o r 20 min a t room t e m p e r a t u r e and t h e n soaked f o r 4 0 min i n 10 m l of 0.1 M Tr is- ma le a t e ,
pH 7.0,
wi t h t h r e e changes.
The g e l s were ne xt im-
mersed i n 1 0 m l of 5% (NH ) S f o r 5 min and t h e n s t o r e d i n w a te r. 4 2 A l k a l i n e p h o sp h a ta se a c t i v i t y appeared a s a brown band. E x t r a c t i o n of enzyme from g e l s by d i f f u s i o n
-
Yeast glycogen s ynthe -
t a s e and E. c o l i a l k a l i n e phosphat ase were e l u t e d from g e l s l i c e s by minci n g t h e g e l wi th a c l e a n r a z o r b l a d e , t h e n t r i t u r a t i n g w i t h a g l a s s rod or by homogenizing i n a t i s s u e homogenizer, r e s p e c t i v e l y .
Tris-buffered
30%
499
PROTEINS FROM POLYACRYLAMIDE GELS g l y c e r o l was added t o t h e g e l p a r t i c l e s which were t h e n removed by c e n t r i f u g a t i o n f o r 10 min a t 10,000 x g. A n a l y t i c a l procedures
- Alkaline
p h o s p h a t a s e 1 2 and y e a s t g l y c o g e n
s y n t h e t a s e 1 3 were a s s a y e d a s p r e v i o u s l y d e s c r i b e d . t h e p r o c e d u r e of Warburg and C h r i s t i a n .
P r o t e i n was d e t e r m i n e d by
14
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RESULTS To i l l u s t r a t e t h e e f f i c i e n c y of t h i s p r o c e d u r e , a commercial p r e p a r a -
t i o n of E. c o l i a l k a l i n e p h o s p h a t a s e was s e p a r a t e d on 5 g e l s . d e m o n s t r a t e s t h e h e t e r o g e n e i t y of t h i s enzyme p r e p a r a t i o n .
Fig. 2 , g e l b l ,
Gels # 2 , 4 , 6 ,
and 8 were s e p a r a t e d i n p a r a l l e l and t h e segment of each g e l which c o r r e s p o n d e d t o t h e major p r o t e i n band i n g e l # I was s e c t i o n e d and e l e c t r o p h o r e t i c a l l y e l u t e d a s d e s c r i b e d i n "Methods."
The e l u a t e s , on r e e l e c t r o p h o r e s i s i n a n a l y -
t i c a l g e l s , produced a s i n g l e band ( F i g . 2 , g e l s 6 3 , 5 , 7 , and 9 ) .
The remains
of t h e o r i g i n a l g e l s were s t a i n e d and t h e s e showed t h e p r e s e n c e of t h e c o n t a m i n a n t s ( F i g . 2 , g e l s # 2 , 4 , 6 , and 8 ) .
The r e c o v e r y of enzymatic a c t i v i t y i n
t h i s experiment v a r i e d w i t h t h e d u r a t i o n of r e v e r s e d e l e c t r o p h o r e s i s ( T a b l e I ) . Maximal r e c o v e r y appeared t o be o b t a i n e d a f t e r 3.5 h r , whereas a f t e r o v e r n i g h t e l e c t r o p h o r e s i s t h e y i e l d was l e s s .
The r e c o v e r y of o n l y 47.7% of t h e a c t i v i t y
may be a t t r i b u t e d i n p a r t t o t h e f a c t t h a t more t h a n one e n z y m a t i c a l l y a c t i v e component was p r e s e n t in t h e m i x t u r e , as shown i n F i g . 3, and o n l y t h e major component was i s o l a t e d i n t h i s experiment.
When homogeneous enzyme o b t a i n e d
by e l e c t r o p h o r e t i c i s o l a t i o n was used a s t h e a o u r c e of s t a r t i n g m a t e r i a l , a g a i n t h e r e c o v e r y was a f u n c t i o n of r e v e r s e d e l e c t r o p h o r e s i s t i m e , b u t t h e maximum y i e l d was now 86% ( F i g . 4 ) .
'Ihe y i e l d a p p e a r e d t o d e c l i n e
s l i g h t l y a f t e r r e a c h i n g a maximum a t 6 h o u r s .
For comparison i n t h e
same e x p e r i m e n t , a l k a l i n e p h o s p h a t a s e was e l u t e d from homogenized g e l s l i c e s by s i m p l e d i f f u s i o n .
A f t e r 6 h o u r s , o n l y 42% of t h e a c t i v i t y was
recovered i n the b u f f e r . This method h a s a l s o been used s u c c e s s f u l l y a s t h e f i n a l s t e p of p u r i f i c a t i o n of a tumor a n t i g e n from a human lung tumor e x t r a c t . 1 5
The p a r t i a l l y
BRAATZ AND MC INTIRE
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500
p u r i f i e d a n t i g e n was r a d i o l a b e l e d with 1251and t h e n s u b j e c t e d t o g e l e l e c trophoresis.
The r e s u l t of t h i s a n a l y s i s i s shown i n P i g . 5A.
Other
experiments had i n d i c a t e d t h e p r o t e i n band a t about 7.6 cm ( i n d i c a t e d by t h e b a r ) t o be t h e tumor a n t i g e n .
lhis l a b e l e d a n t i g e n was t h e n i s o l a t e d by
r e v e r s e d e l e c t r o p h o r e t i c e l u t i o n from t h e g e l s l i c e s as d e s c r i b e d i n "Methods." Table 11.
The r e s u l t s of e i g h t s e p a r a t e experiments are summarized i n
F i f t y t o 72% of t h e l a b e l e d a n t i g e n could b e r e c o v e r e d a f t e r 3.5
t o 16 h r of e l e c t r o p h o r e s i s i n t h e p r e s e n c e of g l y c e r o l .
Less t h a n 10% of
t h e r a d i o a c t i v i t y i n t h e g e l s l i c e s a t t h e s t a r t of t h e experiment remained
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PROTEINS FROM POLYACRYLAMIDE GELS
501
(b)
FIGURE 1 Ap p a r a tu s f o r i s o l a t i o n of p r o t e i n from a c r y l a m i d e g e l s . A. 7% acr y la mi d e s u p p o r t g e l (40 mm g e l i n a 6 x 7 5 mm t u b e ) , on t o p of which h a s Dialysis been p l a c e d s e v e r a l 2 mm g e l s l i c e s and e l e c t r o p h o r e s i s b u f f e r . t u b i n g i s a t t a c h e d , f i l l e d wi t h e l e c t r o p h o r e s i s b u f f e r , s e c u r e d w i t h a B. F i n a l a rra nge me nt r u b b e r O-ring and t h e n t i g h t l y t i e d o f f a t t h e top. o f s u p p o r t g e l and g e l s l i c e s i n t h e e l e c t r o p h o r e s i s chamber. The anode i s t h e upper chamber. w i t h i n t h e s l i c e s r e g a r d l e s s of t h e d u r a t i o n of e l e c t r o p h o r e s i s .
Some
r a d i o a c t i v i t y was found a s s o c i a t e d w i t h t h e empty d i a l y s i s sacs ( a p p r o x i m ate l y 8% o f t h e cpm p r e s e n t i n t h e g e l s l i c e s a t t h e s t a r t ) , a s w e l l a s i n t h e upper chamber e l e c t r o p h o r e s i s b u f f e r , which e n a b l e d a p p r o x i m a t e l y 85-90%
of t h e i n i t i a l r a d i o a c t i v i t y t o be account ed f o r .
FIGURE 2. Polyacrylamide gel electrophoresis of E. coli alkaline phosphatase. Gels #I, 2 , 4. 6 , and 8 were electrophoresed at 4 O C . G e l 81 contained 24 g of phosphatase and was stained for protein. Forty g was applied to 1 2 , 4, 6, and 8, from which the region corraponding to the major protein band in gel 111 was sectioned and the protein electrophoretically eluted. The regions which were sectioned are shown here as gaps in gels 82. 4 , 6 , and 8 . The upper and lower segments of these gels are shown after sectioning and stained with Coomassie Blue. Gels 83, 5 , 7, and 9 are analyses of the protein isolated from gels 82, 4 , 6 , and 8. respectively. A small piece of wire marked the position of the tracking dye.
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2
5
Ei
VI 0 N
PROTEINS FROM POLYACRYLAMLDE GELS
503 TABLE I
Recovery of a l k a l i n e p h o s p h a t a s e from g e l s l i c e s by r e v e r s e d e l e c t r o p h o r e s i s
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Hours of reversed electrophoresis
'A
Volume of eluate, ml
A l k a l i n e phosphatase activity (units) i n control' sample
eluate
Yield %
1.5
1.3
0.864
0.178
20.6
3.5
1.13
0.820
0.391
47.7
19.5
1.07
0.800
0.173
21.6
22.5
1.44
0.778
0.111
14.3
b
p o r t i o n of t h e enzyme s o l u t i o n was k e p t a t t h e same t e m p e r a t u r e f o r t h e
d u r a t i o n of t h e e x p e r i m e n t , and a s s a y e d a l o n g w i t h e a c h e l u a t e a t t h e
times i n d i c a t e d . b Y i e l d s a r e based on t h e a c t i v i t i e s r e m a i n i n g i n t h e c o n t r o l sample.
The r e c o v e r y of r a d i o l a b e l e d p r o t e i n i n t h e d i a l y s i s s a c was enhanced by t h e p r e s e n c e of 30% g l y c e r o l i n t h e e l e c t r o p h o r e s i s b u f f e r ( T a b l e 11).
Thus i n t h e p r e s e n c e of g l y c e r o l t h e mean y i e l d was 57.6 L 7.5%; i n t h e a b s e n c e of g l y c e r o l a mean of 32.0
L 12.0% was r e c o v e r e d .
The l a b e l e d
p r o t e i n r e c o v e r e d by t h i s p r o c e d u r e e x h i b i t e d a s i n g l e band on d i s c g e l ( F i g . 5B). I n a f u r t h e r e x p e r i m e n t , y e a s t glycogen s y n t h e t a s e was i s o l a t e d from p o l y a c r y l a m i d e g e l s l i c e s a f t e r a n o v e r n i g h t (16 h r ) e l e c t r o p h o r e s i s .
As
shown i n T a b l e 111, 73% o f t h e enzymatic a c t i v i t y was r e c o v e r e d by e l e c t r o p h o r e s i s a s compared t o o n l y 12% by d i f f u s i o n .
DISCUSSION ' h i s paper d e s c r i b e s a method f o r i s o l a t i n g p r o t e i n s from p o l y a c r y l a mide g e l s which i s a ) s i m p l e , s i n c e i t r e q u i r e s s t a n d a r d a n a l y t i c a l g e l e l e c t r o p h o r e s i s equipment w i t h o u t m o d i f i c a t i o n , b) r a p i d , s i n c e maximal
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FIGURE 3 Disc g e l e l e c t r o p h o r e s i s of E. c o l i a l k a l i n e phosphatase, s t a i n e d for enzymatic a c t i v i t y . Ihirty-seven Ug of enzyme were applied and e l e c t r o phoresis was carried out a t 4'C and s t a i n e d a s described i n "Methods." Enzymatic a c t i v i t y appeared a s a brown p r e c i p i t a t e of PbS.
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PROTEINS FROM POLYACRYLAMIDE GELS
505
HOURS FIGURE 4 Recovery of enzymatic a c t i v i t y from g e l s l i c e s by e l e c t r o p h o r e t i c e l u t i o n . A p o o l of a c r y l a m i d e g e l - p u r i f i e d a l k a l i n e p h o s p h a t a s e from g e l s # 2 , 4 , 6 , and 8 , Fig. 2 , was e l e c t r o p h o r e s e d on 4 g e l s . I h e g e l r e g i o n s c o r r e s p o n d i n g t o t h e p r o t e i n band i n a f i f t h ( m a r k e r ) g e l were e l u t e d a s b e f o r e i n t o a f i n a l volume of less t h a n 1.6 m l . Twenty-five ug of enzyme (0.0815 u n i t s ) were a p p l i e d t o each g e l .
r e c o v e r y is o b t a i n e d i n 4-6 h r , and c ) e f f i c i e n t , p e r m i t t i n g y i e l d s of up t o 86% i n a homogeneous form.
The e n t i r e p r o c e d u r e c a n be performed i n
one day and would t h e r e f o r e be u s e f u l i n t h e i s o l a t i o n of l a b i l e p r o t e i n s . F u r t h e r m o r e , t h e f i n a l volume of t h e e l u a t e c a n be k e p t s m a l l ( e a s i l y less t h a n 1.5 ml) t h u s o b v i a t i n g t h e need f o r f u r t h e r c o n c e n t r a t i o n which may lead to a d d i t i o n a l losses.
'Ihe method c a n be used w i t h v e r y small amounts
of p r o t e i n , a s h a s been shown w i t h t r a c e r q u a n t i t i e s of 1251-labeled
tumor
a n t i g e n , o r w i t h l a r g e r amounts of p r o t e i n by r u n n i n g a series of r e s o l v i n g gels.
T h i s e n t i r e p r o c e d u r e c o u l d be a p p l i e d t o g e l s of l a r g e r d i a m e t e r
which would p e r m i t working w i t h s t i l l l a r g e r q u a n t i t i e s of p r o t e i n . 'Ihe i n c l u s i o n of 30% g l y c e r o l h a s been found t o i n c r e a s e t h e s t a b i l i t y of c e r t a i n enzymes."
I t h a s t h e r e f o r e been used h e r e and i t a p p e a r s from
506
BRAATZ AND MC I N T I R E
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I. A.
cm
ll ;" 'L
2t 0
1
2
3
4
5
cm FIGURE 5 Polyacr) d d e gel electrophoresis of radioiodinated lung tumor antigen preparation. A. Partially purified iodinated antigen electrophoresed on an 1 1 cm gel. Electrophoresis was continued for one hour after the tracking dye reached the bottom of the gel. Two mm slices were counted in a gamma counter and the slices containing the band indicated by the bar were subjected to reversed electrophoresis. B. The isolated antigen was re-electrophoresed on a shorter, analytical gel, sliced and counted as before.
Table I1 to be beneficial since the yields were consistently higher in the presence of glycerol. lhe average recovery of labeled protein in Table 11, experiments 1-5, was 57.6 5 7.5%.
lhis figure is an underestimate of the true recovery
obtained, since the value on which the yield is based (Table 11, cpm in gel slices before reversed electrophoresis) contained a significant
Glycerola
816,843 270,396
706,000 697,824
103,802 12,263 56,577 142,443 n.d.c 375,732
1,494,240 374,409 476,985 1,289,828 1,402,808 4,271,718 2,732,324 791,934
3.5
4.0
4.5
4.5
16.0
4.0
4.5
16.0
1.1
676,280 175,000
61,458 n.d.c
‘n.d..
not determined.
b . Y i e l d = cpm i n e l u a t e a f t e r d i a l y s i s / c p m i n g e l s l i c e s b e f o r e r e v e r s e d e l e c t r o p h o r e s i s x 100.
2.24
1.9
3.0
1.02
1.3
1.48
1.48
ml
Eluate volume
2,081,640
264,810
eluate
gel slices a f t e r reversed eleCtKOphOKeSiS
cpm i n g e l s l i c e s before reversed eleCtKOphOKeSiS
Hours of reversed electrophoresis
a30% g l y c e r o l was added t o t h e e l e c t r o p h o r e s i s b u f f e r i n b o t h chambers where i n d i c a t e d .
Experiment
Recovery of 1 2 5 1 - l a b e l e d p r o t e i n from g e l s l i c e s by r e v e r s e d e l e c t r o p h o r e s i s
TABLE I1
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22
25
49
50
55
56
72
55
I
Yield
b
50 8
BRAATZ AND MC INTIRE TABLE 111 Comparison of d i f f u s i o n and e l e c t r o p h o r e t i c methods f o r e l u t i n g y e a s t glycogen s y n t h e t a s e from polyacrylamide g e l s l i c e s
Time
of eluate
(hr)
(ml)
VOl.
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Method
Total a c t i v i t y (units) applied
recovered
%
Diffusion
20.5
6.5
1.35
0.162
12
Electrophoresis
16.0
1.9
1.14
0.83
73
A l l b u f f e r s i n t h i s experiment c o n t a i n e d 1 mM 2-mercaptoethanol
and 30%
glycerol.
amount of low m o l e c u l a r weight lZ5I, which i s e v i d e n t from t h e high background of r a d i o a c t i v i t y i n Fig. 5.
I n f a c t , t h e r a d i o l a b e l e d samples
i n i t i a l l y a p p l i e d t o t h e g e l s were o n l y about 70% p r e c i p i t a b l e w i t h 10% TCA. Even a f t e r e x t e n s i v e d i a l y s i s t h i s f i g u r e was d i f f i c u l t t o improve upon and may be due t o l i b e r a t i o n of r a d i o i o d i d e from t h e p r o t e i n a n t i g e n s .
17
Maximal r e c o v e r i e s were o b t a i n e d a f t e r 4-6 h r of r e v e r s e d e l e c t r o phoresis.
With a l k a l i n e phosphatase, t h e y i e l d d e c r e a s e d on prolonged
electrophoresis.
However, t h e p u r i f i e d p r e p a r a t i o n s of lung a n t i g e n (Table
11) r e a c t e d t o t h e same e x t e n t with s p e c i f i c a n t i b o d y r e g a r d l e s s of t h e
t i m e of e l e c t r o p h o r e s i s .
I n a s e p a r a t e experiment, 13% of y e a s t glycogen
s y n t h e t a s e a c t i v i t y was recovered a f t e r 16 h r of e l e c t r o p h o r e s i s .
mere-
f o r e , t h e loss of a c t i v i t y as found with a l k a l i n e phosphataae does n o t appear t o be a g e n e r a l phenomenon. m e procedure d e s c r i b e d i n t h i s paper should b e a p p l i c a b l e t o r e c o v e r y of a wide v a r i e t y of p r o t e i n s a f t e r e l e c t r o p h o r e t i c s e p a r a t i o n from cont a m i n a t i n g components.
PROTEINS FROM POLYACRYLAMIDE GELS
509
ACKNOWLEDGMENTS The a u t h o r s g r a t e f u l l y acknowledge t h e e x p e r t t e c h n i c a l a s s i s t a n c e
of Mr. Bryan Boozer.
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REFERENCES
172,440-451 (1971). 121,382-390 (1964).
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A.
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J. V. M a i z e l , Ann. N.Y.
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M. J . Dunn and A. H. Maddy, i n "Biochemical A n a l y s i s of Membrane
Chrambach and D.
Rodbard, S c i e n c e , Acad. S c i . ,
P r o t e i n s , " A. H. Maddy, e d . ,
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D.
S u l i t z e a n u and W.
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D.
S u l i t z e a n u , M.
John Wiley, New York, 1976, pp.
F. Goldman, N a t u r e ,
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197-251.
1120-1121 (1965).
S l a v i n and E. Y e c h e s k e l i , Anal. Biochem.
2,57-67
(1967).
65,
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R. E. S t e p h e n s , Anal. Biochem.,
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H. Wada, H. Kagamiyama, K. M i y a t a k i , Y. Osaka Univ.,
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369-379 (1975). Nomata and H.
Yadao, Med. J.
25, 125-140 (1975).
K. P. Huang and E. Cabib, Biochem. Biophys. Res. Commun.,
69,
1610-1616
(1972). 9. 10.
B. J. Davis, Ann. N.Y.
Acad.
A b b r e v i a t i o n s used a r e :
Sci.,
121,404-427
(1964).
TCA, t r i c h l o r o a c e t i c a c i d ; T r i s , T r i s ( h y d r o x y -
m e t h y l ) aminome thane.
(1963).
J. M. A l l e n and G.
12.
R. H. Glew and E. C. Heath, J. B i o l . Chem.,
13.
K. P. Huang and E. Cabib, Biochim. Biophys. A c t a ,
14.
0. Warburg and W.
15.
J. A. B r a a t z , K. R. M c I n t i r e , G. L. P r i n c l e r , K. H. K o r t r i g h t and R.
16.
B.
S. L.
Hyncik, J. Histochem. Cytochem.,
11,169-
11.
246, 1556-1565 (1971).
C h r i s t i a n , Methods Enzymol.,
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302, 240-248 (1973). 454-456 (1957).
Herberman, s u b m i t t e d f o r p u b l i c a t i o n . Bradbury and W.
B. Jakoby, P r o c . Nat.
Acad. S c i . ,
69,
2373-2376
(1972). 17.
L. R. Chevru and D. R. K. Murty, i n "Radioimmunoassay," and M. D.
Blaufox, e d s . ,
L. M. Freeman
Grune and S t r a t t o n , New York, 1975, pp. 33-48.
Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
A Rapid and Efficient Method for the Isolation of Proteins from Polyacrylamide Gels a
J. A. Braatzw & K. R. McLntire
a
a
Laboratory of Immunodiagnosis , National Cancer Institute, National Institutes of Health , Bethesda, Maryland, 20014 Published online: 05 Mar 2007.
To cite this article: J. A. Braatzw & K. R. McLntire (1977) A Rapid and Efficient Method for the Isolation of Proteins from Polyacrylamide Gels, Preparative Biochemistry, 7:6, 495-509, DOI: 10.1080/00327487708065516 To link to this article: http://dx.doi.org/10.1080/00327487708065516
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PREPARATIVE BIOCHEMISTRY, 7 ( 6 ) , 495-509
(1977)
A RAPID AND EFFICIENT METHOD FOR THE ISOLATION OF PROTEINS FROM POLYACRYLAMIDE GELS
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J. A. Braat z and K. R. M c I n t i r e L a b o r a t o r y of Immunodiagnosis, N a t i o n a l Cancer I n s t i t u t e , N a t i o n a l I n s t i t u t e s of H e a l t h , Bet hesda, Maryland 20014 ABSTRACT A p r o c e d u r e i s d e s c r i b e d f o r t h e r a p i d and e f f i c i e n t e l e c t r o p h o r e t i c
e l u t i o n o f p r o t e i n from pol yacryl ami de g e l s which i s t h e n c o l l e c t e d i n a d i a l y s i s bag t i e d t o t h e end of a t u b e c o n t a i n i n g t h e g e l s l i c e s .
To
i l l u s t r a t e t h e method a het erogeneous p r e p a r a t i o n of a l k a l i n e phos pha ta s e was used from which a s i n g l e homogeneous component was i s o l a t e d i n s i x hours w i t h a r e c o v e r y of 86%.
The e l u t e d p r o t e i n i s c o l l e c t e d i n a volume which
can e a s i l y be k e p t below 1.5 m l , t h u s e l i m i n a t i n g t h e need f o r s ubs e que nt concentration.
The method h a s a l s o been used s u c c e s s f u l l y i n two o t h e r
systems i n which a human lung t umor-associ at ed a n t i g e n and glyc oge n synt h e t a s e from y e a s t were i s o l a t e d .
S i n c e t h e method u t i l i z e s a s t a n d a r d
a n a l y t i c a l g e l e l e c t r o p h o r e s i s a p p a r a t u s w i t h no m o d i f i c a t i o n s or a c c e s s o r i e s , i t sh o u ld be immediately a p p l i c a b l e f o r t h e i s o l a t i o n of many d i f f e r e n t p r o t e i n s from pol yacryl ami de g e l s . INTRODUCTION A n a l y t i c a l p o lyacryl ami de g e l e l e c t r o p h o r e s i s is one of t h e most powerful methods a v a i l a b l e f o r d e t e r m i n i n g t h e comple xity or p u r i t y of p r o t e i n Solutions.'
F r e q u e n t l y , however, i t becomes n e c e s s a r y t o go beyond
t h e a n a l y t i c a l s t a g e by a t t e m p t i n g t o i s o l a t e s p e c i f i c p r o t e i n bands from the gel.
For example, t h e d e m o n s t r a t i o n of enzymatic o r a n t i g e n i c a c t i v i t y
a s s o c i a t e d w i t h a p a r t i c u l a r band may r e q u i r e t h e e l u t i o n and r e c o v e r y of
495 Copyright 8 1977 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any !nformatlon storageand retrieval system. without permission in writing from the publisher.
B W T Z AND MC INTIRE
49 6 that component from the gel.
Whether for this purpose or for the prepara-
tive isolation of protein from acrylamide gels, the most commonly used procedures have been a) homogenization of the gel region containing the protein followed by extraction with buffer or b) the use of commercially available preparative gel electrophoresis
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Various preparative gel systems are available which are useful for protein purification from relatively large amounts of starting material. If the amount of protein is small, however, it may be lost i n these preparative-scale gel systems due to non-specific adsorption.
On the other
hand, extraction of protein from homogenized gel slices often results in poor recoveries and usually requires long periods for diffusion, during which inactivation or degradation can occur. A number of reports have appeared which describe the electrophoretic elution of protein from polyacrylamide fied gel electrophoresis equipment.
gel^^-^
but these all require modi-
This paper describes a simple procedure
for the electrophoretic elution of proteins from gels which makes use of standard analytical gel electrophoresis equipment. MATERIALS AND METHODS Materials
-
Acrylamide, 99+%, electrophoresis grade and N,N'-Methylene-
bisacrylamide, 99+%, electrophoresis grade were purchased from the Aldrich Chemical Co. Ammonium persulfate, Ultra Pure, was obtained from Polysciences, Inc., and N,N,N',N'-Tetramethylethylenediamine
from Eastman
Kodak Co. Coomassie Brilliant Blue 6 2 5 0 was purchased from EC Apparatus Corp. E. coli alkaline phosphatase Type 111 was obtained from Sigma. Glycogen synthetase was partially purified from yeast as previously described.'
The electrophoresis chamber was obtained from Buchler Instruments
and the power supply was a Beckman Spinco Duostat.
6 nrm i.d.
x 8 mm 0.d.
7 m m 0.d.
x 125 mm from BioRad.
Gel tubes were
x 7 5 mm from Scientific Products, and 5.5 m m i.d.
Preparation of gels
-
x
Standard pH 8.9, 7% acrylamide gels were pre-
pared according to Davis9 using ammonium persulfate to initiate p o l p e r i -
49 7
PROTEINS FROM POLYACRYLAMIDE GELS zation.
The g e l s were r o u t i n e l y c a s t t o a h e i g h t of 60 or 110 mm i n t h e 75
or 125 mm t u b e s , r e s p e c t i v e l y , a t l e a s t one day p r i o r t o u s e i n o r d e r t o a l l o w S t a c k i n g g e l s were omitte d.
t r a c e s of p e r s u l f a t e t o decompose. Electrophoresis
- A maximum volume
of 150 ~1 of sample was mixed w i t h
50 ~1 of g l y c e r o l and 3 ~1of 0.05% Bromphenol Blue and a p p l i e d t o t h e g e l .
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E l e c t r o p h o r e s i s was performed a t 4°C a t a c u r r e n t of 2 ma /tube u n t i l t h e
I n some e x p e r i -
t r a c k i n g dye was about 1 cm from t h e bottom of t h e g e l .
ments i n o r d e r t o o b t a i n b e t t e r band s e p a r s t i o n , e l e c t r o p h o r e s i s was performed on g e l s 110 mm i n l e n g t h and c o n t i n u e d for one hour a f t e r t h e t r a c k i n g dye was r un o f f t h e bottom.
Gels were removed by rimming w i t h a
2 i n c h , 20 gauge n e e d l e while i n j e c t i n g water and t h e p o s i t i o n of t h e In order t o localize
t r a c k i n g dye was marked by i n s e r t i n g a p i e c e of w ire .
t h e p r o t e i n bands, a s e p a r a t e g e l was f i x e d i n 12.5% TCA"
f o r 30 min and
s t a i n e d i n Coomassie B r i l l i a n t Blue (1:20 d i l u t i o n of a 1%aqueous s o l u t i o n wi th 12.5% TCA) f o r 30 min.
P r o t e i n bands c o n t a i n i n g a s l i t t l e as
1 0 ug of p r o t e i n c oul d be v i s u a l i z e d by t h i s r a p i d s t a i n i n g proc e dure which r e q u i r e d no d e s t a i n i n g . k e p t a t -3O'C
The g e l s c o n t a i n i n g t h e p r o t e i n t o be e l u t e d were
and t h e n a l i g n e d w i t h t h e s t a i n e d g e l .
The a p p r o p r i a t e g e l
s e c t i o n s were c u t o u t w i t h a c l e a n r a z o r and t h e p r o t e i n e l u t e d as desc r i b e d below.
Ge l s c o n t a i n i n g r a d i o l a b e l e d p r o t e i n were f r o z e n f o r one
h o u r a t -3O"C,
t h e n s l i c e d i n t o 2 mm s e c t i o n s and c ounte d i n a Beckman
BioGamma c o u n t e r .
The g e l s l i c e s c o n t a i n i n g t h e r a d i o a c t i v e t r a c e of
i n t e r e s t were t h e n s e l e c t e d and t h e p r o t e i n e l u t e d as d e s c r i b e d below. E l e c t r o p h o r e t i c e l u t i o n of p r o t e i n from g e l s l i c e s
- Acrylamide
gels
were c a s t i n t o 75 mm g l a s s t u b e s t o a h e i g h t of 40 mm, i n t h e re ma inde r o f t h e t u b e were p laced t h e g e l s l i c e s c o n t a i n i n g t h e p r o t e i n t o b e e l u t e d . The t u b e s were f i l l e d w i t h e l e c t r o p h o r e s i s b u f f e r (0.025 M T r i s , 0.2 M g l y c i n e , pH 8.3, wi t h or w i t h o u t 30% g l y c e r o l ) and t h e s l i c e s were s t a c k e d on t o p of t h e s u p p o r t g e l , c a r e bei ng t a k e n t o e l i m i n a t e a l l a i r bubble s . D i a l y s i s t u b i n g (Union C arbi de Corp.),
0.5 i n . wide, was a p p l i e d t o t h e
BRAATZ AND MC INTIRE
498 t o p of t h e tube and f i x e d i n p l a c e w i t h a 5 mm i .d.
rubbe r O-ring.
Elec-
t r o p h o r e s i s b u f f e r was added c a r e f u l l y t o avoi d a i r bubble s u n t i l t h e volume o f b u f f e r i n s i d e t h e d i a l y s i s bag p l u s t h a t c o v e r i n g t h e g e l s l i c e s was ab o u t 1 m l .
The t o p of t h e bag was t i e d s e c u r e l y w i t h s t r i n g (Fig. 1A).
The g l a s s t u b e was i n s e r t e d i n t o t h e e l e c t r o p h o r e s i s chamber which was t h e n f i l l e d with buffer.
The f i n a l arrangement is shown i n Fig. 1 B .
It was
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n e c e s s a r y a t t h i s s t a g e t o r e v e r s e t h e e l e c t r o d e s so t h a t t h e p r o t e i n s m igr a t e d toward t h e upper (anode) chamber.
Reversed e l e c t r o p h o r e s i s was
t h e n c a r r i e d o u t a t 4OC a t 2 ms/ gel f o r v a r y i n g t i m e p e r i o d s a f t e r which t h e t u b e s were removed and i n v e r t e d , and t h e d i a l y s i s bag was t h e n c a r e f u l l y removed.
The b u f f e r remaining i n t h e g l a s s column w a s added t o t h e c o n t e n t s
o f t h e d i a l y s i s bag which c a n be t i e d o f f a t t h e o p p o s i t e end and d i a l y z e d i f desired.
I n some experi ment s i t was found c o n v e n i e n t , b u t not n e c e s s a r y ,
t o run t h e f i r s t ( r e s o l v i n g ) e l e c t r o p h o r e s i s i n 5.5 mm diam. g e l s and t h e r e v e r s e d e l e c t r o p h o r e s i s i n g e l t u b e s of 6 mm i.d.
i n order t o f a c i l i t a t e
s t a c k i n g t h e g e l s l i c e s on t op of t h e s u p p o r t g e l . D e t e c t i o n of enzyme a c t i v i t y on g e l s
- Alkaline
phos pha ta s e a c t i v i t y
was d e t e c t e d on g e l s usi ng a m o d i f i c a t i o n of t h e proc e dure of A lle n and Hyncik."
A f t e r e l e c t r o p h o r e s i s g e l s were immersed i n 10 m l of 0.5 M T r i s ,
pH 9.0, c o n t a i n i n g 1 mM p-ni t rophenyl phosphat e and 0.2 M CaCl a t room te mp e r a tu r e .
2
f o r 10 min
The g e l s were t h e n r i n s e d w i t h d i s t i l l e d water and
immersed i n 10 m l of 0.1 M T ri s-mal eat e,
pH 7.0,
c o n t a i n i n g 3 d Pb(N03)2
f o r 20 min a t room t e m p e r a t u r e and t h e n soaked f o r 4 0 min i n 10 m l of 0.1 M Tr is- ma le a t e ,
pH 7.0,
wi t h t h r e e changes.
The g e l s were ne xt im-
mersed i n 1 0 m l of 5% (NH ) S f o r 5 min and t h e n s t o r e d i n w a te r. 4 2 A l k a l i n e p h o sp h a ta se a c t i v i t y appeared a s a brown band. E x t r a c t i o n of enzyme from g e l s by d i f f u s i o n
-
Yeast glycogen s ynthe -
t a s e and E. c o l i a l k a l i n e phosphat ase were e l u t e d from g e l s l i c e s by minci n g t h e g e l wi th a c l e a n r a z o r b l a d e , t h e n t r i t u r a t i n g w i t h a g l a s s rod or by homogenizing i n a t i s s u e homogenizer, r e s p e c t i v e l y .
Tris-buffered
30%
499
PROTEINS FROM POLYACRYLAMIDE GELS g l y c e r o l was added t o t h e g e l p a r t i c l e s which were t h e n removed by c e n t r i f u g a t i o n f o r 10 min a t 10,000 x g. A n a l y t i c a l procedures
- Alkaline
p h o s p h a t a s e 1 2 and y e a s t g l y c o g e n
s y n t h e t a s e 1 3 were a s s a y e d a s p r e v i o u s l y d e s c r i b e d . t h e p r o c e d u r e of Warburg and C h r i s t i a n .
P r o t e i n was d e t e r m i n e d by
14
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RESULTS To i l l u s t r a t e t h e e f f i c i e n c y of t h i s p r o c e d u r e , a commercial p r e p a r a -
t i o n of E. c o l i a l k a l i n e p h o s p h a t a s e was s e p a r a t e d on 5 g e l s . d e m o n s t r a t e s t h e h e t e r o g e n e i t y of t h i s enzyme p r e p a r a t i o n .
Fig. 2 , g e l b l ,
Gels # 2 , 4 , 6 ,
and 8 were s e p a r a t e d i n p a r a l l e l and t h e segment of each g e l which c o r r e s p o n d e d t o t h e major p r o t e i n band i n g e l # I was s e c t i o n e d and e l e c t r o p h o r e t i c a l l y e l u t e d a s d e s c r i b e d i n "Methods."
The e l u a t e s , on r e e l e c t r o p h o r e s i s i n a n a l y -
t i c a l g e l s , produced a s i n g l e band ( F i g . 2 , g e l s 6 3 , 5 , 7 , and 9 ) .
The remains
of t h e o r i g i n a l g e l s were s t a i n e d and t h e s e showed t h e p r e s e n c e of t h e c o n t a m i n a n t s ( F i g . 2 , g e l s # 2 , 4 , 6 , and 8 ) .
The r e c o v e r y of enzymatic a c t i v i t y i n
t h i s experiment v a r i e d w i t h t h e d u r a t i o n of r e v e r s e d e l e c t r o p h o r e s i s ( T a b l e I ) . Maximal r e c o v e r y appeared t o be o b t a i n e d a f t e r 3.5 h r , whereas a f t e r o v e r n i g h t e l e c t r o p h o r e s i s t h e y i e l d was l e s s .
The r e c o v e r y of o n l y 47.7% of t h e a c t i v i t y
may be a t t r i b u t e d i n p a r t t o t h e f a c t t h a t more t h a n one e n z y m a t i c a l l y a c t i v e component was p r e s e n t in t h e m i x t u r e , as shown i n F i g . 3, and o n l y t h e major component was i s o l a t e d i n t h i s experiment.
When homogeneous enzyme o b t a i n e d
by e l e c t r o p h o r e t i c i s o l a t i o n was used a s t h e a o u r c e of s t a r t i n g m a t e r i a l , a g a i n t h e r e c o v e r y was a f u n c t i o n of r e v e r s e d e l e c t r o p h o r e s i s t i m e , b u t t h e maximum y i e l d was now 86% ( F i g . 4 ) .
'Ihe y i e l d a p p e a r e d t o d e c l i n e
s l i g h t l y a f t e r r e a c h i n g a maximum a t 6 h o u r s .
For comparison i n t h e
same e x p e r i m e n t , a l k a l i n e p h o s p h a t a s e was e l u t e d from homogenized g e l s l i c e s by s i m p l e d i f f u s i o n .
A f t e r 6 h o u r s , o n l y 42% of t h e a c t i v i t y was
recovered i n the b u f f e r . This method h a s a l s o been used s u c c e s s f u l l y a s t h e f i n a l s t e p of p u r i f i c a t i o n of a tumor a n t i g e n from a human lung tumor e x t r a c t . 1 5
The p a r t i a l l y
BRAATZ AND MC INTIRE
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500
p u r i f i e d a n t i g e n was r a d i o l a b e l e d with 1251and t h e n s u b j e c t e d t o g e l e l e c trophoresis.
The r e s u l t of t h i s a n a l y s i s i s shown i n P i g . 5A.
Other
experiments had i n d i c a t e d t h e p r o t e i n band a t about 7.6 cm ( i n d i c a t e d by t h e b a r ) t o be t h e tumor a n t i g e n .
lhis l a b e l e d a n t i g e n was t h e n i s o l a t e d by
r e v e r s e d e l e c t r o p h o r e t i c e l u t i o n from t h e g e l s l i c e s as d e s c r i b e d i n "Methods." Table 11.
The r e s u l t s of e i g h t s e p a r a t e experiments are summarized i n
F i f t y t o 72% of t h e l a b e l e d a n t i g e n could b e r e c o v e r e d a f t e r 3.5
t o 16 h r of e l e c t r o p h o r e s i s i n t h e p r e s e n c e of g l y c e r o l .
Less t h a n 10% of
t h e r a d i o a c t i v i t y i n t h e g e l s l i c e s a t t h e s t a r t of t h e experiment remained
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PROTEINS FROM POLYACRYLAMIDE GELS
501
(b)
FIGURE 1 Ap p a r a tu s f o r i s o l a t i o n of p r o t e i n from a c r y l a m i d e g e l s . A. 7% acr y la mi d e s u p p o r t g e l (40 mm g e l i n a 6 x 7 5 mm t u b e ) , on t o p of which h a s Dialysis been p l a c e d s e v e r a l 2 mm g e l s l i c e s and e l e c t r o p h o r e s i s b u f f e r . t u b i n g i s a t t a c h e d , f i l l e d wi t h e l e c t r o p h o r e s i s b u f f e r , s e c u r e d w i t h a B. F i n a l a rra nge me nt r u b b e r O-ring and t h e n t i g h t l y t i e d o f f a t t h e top. o f s u p p o r t g e l and g e l s l i c e s i n t h e e l e c t r o p h o r e s i s chamber. The anode i s t h e upper chamber. w i t h i n t h e s l i c e s r e g a r d l e s s of t h e d u r a t i o n of e l e c t r o p h o r e s i s .
Some
r a d i o a c t i v i t y was found a s s o c i a t e d w i t h t h e empty d i a l y s i s sacs ( a p p r o x i m ate l y 8% o f t h e cpm p r e s e n t i n t h e g e l s l i c e s a t t h e s t a r t ) , a s w e l l a s i n t h e upper chamber e l e c t r o p h o r e s i s b u f f e r , which e n a b l e d a p p r o x i m a t e l y 85-90%
of t h e i n i t i a l r a d i o a c t i v i t y t o be account ed f o r .
FIGURE 2. Polyacrylamide gel electrophoresis of E. coli alkaline phosphatase. Gels #I, 2 , 4. 6 , and 8 were electrophoresed at 4 O C . G e l 81 contained 24 g of phosphatase and was stained for protein. Forty g was applied to 1 2 , 4, 6, and 8, from which the region corraponding to the major protein band in gel 111 was sectioned and the protein electrophoretically eluted. The regions which were sectioned are shown here as gaps in gels 82. 4 , 6 , and 8 . The upper and lower segments of these gels are shown after sectioning and stained with Coomassie Blue. Gels 83, 5 , 7, and 9 are analyses of the protein isolated from gels 82, 4 , 6 , and 8. respectively. A small piece of wire marked the position of the tracking dye.
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2
5
Ei
VI 0 N
PROTEINS FROM POLYACRYLAMLDE GELS
503 TABLE I
Recovery of a l k a l i n e p h o s p h a t a s e from g e l s l i c e s by r e v e r s e d e l e c t r o p h o r e s i s
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Hours of reversed electrophoresis
'A
Volume of eluate, ml
A l k a l i n e phosphatase activity (units) i n control' sample
eluate
Yield %
1.5
1.3
0.864
0.178
20.6
3.5
1.13
0.820
0.391
47.7
19.5
1.07
0.800
0.173
21.6
22.5
1.44
0.778
0.111
14.3
b
p o r t i o n of t h e enzyme s o l u t i o n was k e p t a t t h e same t e m p e r a t u r e f o r t h e
d u r a t i o n of t h e e x p e r i m e n t , and a s s a y e d a l o n g w i t h e a c h e l u a t e a t t h e
times i n d i c a t e d . b Y i e l d s a r e based on t h e a c t i v i t i e s r e m a i n i n g i n t h e c o n t r o l sample.
The r e c o v e r y of r a d i o l a b e l e d p r o t e i n i n t h e d i a l y s i s s a c was enhanced by t h e p r e s e n c e of 30% g l y c e r o l i n t h e e l e c t r o p h o r e s i s b u f f e r ( T a b l e 11).
Thus i n t h e p r e s e n c e of g l y c e r o l t h e mean y i e l d was 57.6 L 7.5%; i n t h e a b s e n c e of g l y c e r o l a mean of 32.0
L 12.0% was r e c o v e r e d .
The l a b e l e d
p r o t e i n r e c o v e r e d by t h i s p r o c e d u r e e x h i b i t e d a s i n g l e band on d i s c g e l ( F i g . 5B). I n a f u r t h e r e x p e r i m e n t , y e a s t glycogen s y n t h e t a s e was i s o l a t e d from p o l y a c r y l a m i d e g e l s l i c e s a f t e r a n o v e r n i g h t (16 h r ) e l e c t r o p h o r e s i s .
As
shown i n T a b l e 111, 73% o f t h e enzymatic a c t i v i t y was r e c o v e r e d by e l e c t r o p h o r e s i s a s compared t o o n l y 12% by d i f f u s i o n .
DISCUSSION ' h i s paper d e s c r i b e s a method f o r i s o l a t i n g p r o t e i n s from p o l y a c r y l a mide g e l s which i s a ) s i m p l e , s i n c e i t r e q u i r e s s t a n d a r d a n a l y t i c a l g e l e l e c t r o p h o r e s i s equipment w i t h o u t m o d i f i c a t i o n , b) r a p i d , s i n c e maximal
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FIGURE 3 Disc g e l e l e c t r o p h o r e s i s of E. c o l i a l k a l i n e phosphatase, s t a i n e d for enzymatic a c t i v i t y . Ihirty-seven Ug of enzyme were applied and e l e c t r o phoresis was carried out a t 4'C and s t a i n e d a s described i n "Methods." Enzymatic a c t i v i t y appeared a s a brown p r e c i p i t a t e of PbS.
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PROTEINS FROM POLYACRYLAMIDE GELS
505
HOURS FIGURE 4 Recovery of enzymatic a c t i v i t y from g e l s l i c e s by e l e c t r o p h o r e t i c e l u t i o n . A p o o l of a c r y l a m i d e g e l - p u r i f i e d a l k a l i n e p h o s p h a t a s e from g e l s # 2 , 4 , 6 , and 8 , Fig. 2 , was e l e c t r o p h o r e s e d on 4 g e l s . I h e g e l r e g i o n s c o r r e s p o n d i n g t o t h e p r o t e i n band i n a f i f t h ( m a r k e r ) g e l were e l u t e d a s b e f o r e i n t o a f i n a l volume of less t h a n 1.6 m l . Twenty-five ug of enzyme (0.0815 u n i t s ) were a p p l i e d t o each g e l .
r e c o v e r y is o b t a i n e d i n 4-6 h r , and c ) e f f i c i e n t , p e r m i t t i n g y i e l d s of up t o 86% i n a homogeneous form.
The e n t i r e p r o c e d u r e c a n be performed i n
one day and would t h e r e f o r e be u s e f u l i n t h e i s o l a t i o n of l a b i l e p r o t e i n s . F u r t h e r m o r e , t h e f i n a l volume of t h e e l u a t e c a n be k e p t s m a l l ( e a s i l y less t h a n 1.5 ml) t h u s o b v i a t i n g t h e need f o r f u r t h e r c o n c e n t r a t i o n which may lead to a d d i t i o n a l losses.
'Ihe method c a n be used w i t h v e r y small amounts
of p r o t e i n , a s h a s been shown w i t h t r a c e r q u a n t i t i e s of 1251-labeled
tumor
a n t i g e n , o r w i t h l a r g e r amounts of p r o t e i n by r u n n i n g a series of r e s o l v i n g gels.
T h i s e n t i r e p r o c e d u r e c o u l d be a p p l i e d t o g e l s of l a r g e r d i a m e t e r
which would p e r m i t working w i t h s t i l l l a r g e r q u a n t i t i e s of p r o t e i n . 'Ihe i n c l u s i o n of 30% g l y c e r o l h a s been found t o i n c r e a s e t h e s t a b i l i t y of c e r t a i n enzymes."
I t h a s t h e r e f o r e been used h e r e and i t a p p e a r s from
506
BRAATZ AND MC I N T I R E
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I. A.
cm
ll ;" 'L
2t 0
1
2
3
4
5
cm FIGURE 5 Polyacr) d d e gel electrophoresis of radioiodinated lung tumor antigen preparation. A. Partially purified iodinated antigen electrophoresed on an 1 1 cm gel. Electrophoresis was continued for one hour after the tracking dye reached the bottom of the gel. Two mm slices were counted in a gamma counter and the slices containing the band indicated by the bar were subjected to reversed electrophoresis. B. The isolated antigen was re-electrophoresed on a shorter, analytical gel, sliced and counted as before.
Table I1 to be beneficial since the yields were consistently higher in the presence of glycerol. lhe average recovery of labeled protein in Table 11, experiments 1-5, was 57.6 5 7.5%.
lhis figure is an underestimate of the true recovery
obtained, since the value on which the yield is based (Table 11, cpm in gel slices before reversed electrophoresis) contained a significant
Glycerola
816,843 270,396
706,000 697,824
103,802 12,263 56,577 142,443 n.d.c 375,732
1,494,240 374,409 476,985 1,289,828 1,402,808 4,271,718 2,732,324 791,934
3.5
4.0
4.5
4.5
16.0
4.0
4.5
16.0
1.1
676,280 175,000
61,458 n.d.c
‘n.d..
not determined.
b . Y i e l d = cpm i n e l u a t e a f t e r d i a l y s i s / c p m i n g e l s l i c e s b e f o r e r e v e r s e d e l e c t r o p h o r e s i s x 100.
2.24
1.9
3.0
1.02
1.3
1.48
1.48
ml
Eluate volume
2,081,640
264,810
eluate
gel slices a f t e r reversed eleCtKOphOKeSiS
cpm i n g e l s l i c e s before reversed eleCtKOphOKeSiS
Hours of reversed electrophoresis
a30% g l y c e r o l was added t o t h e e l e c t r o p h o r e s i s b u f f e r i n b o t h chambers where i n d i c a t e d .
Experiment
Recovery of 1 2 5 1 - l a b e l e d p r o t e i n from g e l s l i c e s by r e v e r s e d e l e c t r o p h o r e s i s
TABLE I1
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22
25
49
50
55
56
72
55
I
Yield
b
50 8
BRAATZ AND MC INTIRE TABLE 111 Comparison of d i f f u s i o n and e l e c t r o p h o r e t i c methods f o r e l u t i n g y e a s t glycogen s y n t h e t a s e from polyacrylamide g e l s l i c e s
Time
of eluate
(hr)
(ml)
VOl.
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Method
Total a c t i v i t y (units) applied
recovered
%
Diffusion
20.5
6.5
1.35
0.162
12
Electrophoresis
16.0
1.9
1.14
0.83
73
A l l b u f f e r s i n t h i s experiment c o n t a i n e d 1 mM 2-mercaptoethanol
and 30%
glycerol.
amount of low m o l e c u l a r weight lZ5I, which i s e v i d e n t from t h e high background of r a d i o a c t i v i t y i n Fig. 5.
I n f a c t , t h e r a d i o l a b e l e d samples
i n i t i a l l y a p p l i e d t o t h e g e l s were o n l y about 70% p r e c i p i t a b l e w i t h 10% TCA. Even a f t e r e x t e n s i v e d i a l y s i s t h i s f i g u r e was d i f f i c u l t t o improve upon and may be due t o l i b e r a t i o n of r a d i o i o d i d e from t h e p r o t e i n a n t i g e n s .
17
Maximal r e c o v e r i e s were o b t a i n e d a f t e r 4-6 h r of r e v e r s e d e l e c t r o phoresis.
With a l k a l i n e phosphatase, t h e y i e l d d e c r e a s e d on prolonged
electrophoresis.
However, t h e p u r i f i e d p r e p a r a t i o n s of lung a n t i g e n (Table
11) r e a c t e d t o t h e same e x t e n t with s p e c i f i c a n t i b o d y r e g a r d l e s s of t h e
t i m e of e l e c t r o p h o r e s i s .
I n a s e p a r a t e experiment, 13% of y e a s t glycogen
s y n t h e t a s e a c t i v i t y was recovered a f t e r 16 h r of e l e c t r o p h o r e s i s .
mere-
f o r e , t h e loss of a c t i v i t y as found with a l k a l i n e phosphataae does n o t appear t o be a g e n e r a l phenomenon. m e procedure d e s c r i b e d i n t h i s paper should b e a p p l i c a b l e t o r e c o v e r y of a wide v a r i e t y of p r o t e i n s a f t e r e l e c t r o p h o r e t i c s e p a r a t i o n from cont a m i n a t i n g components.
PROTEINS FROM POLYACRYLAMIDE GELS
509
ACKNOWLEDGMENTS The a u t h o r s g r a t e f u l l y acknowledge t h e e x p e r t t e c h n i c a l a s s i s t a n c e
of Mr. Bryan Boozer.
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REFERENCES
172,440-451 (1971). 121,382-390 (1964).
1.
A.
2.
J. V. M a i z e l , Ann. N.Y.
3.
M. J . Dunn and A. H. Maddy, i n "Biochemical A n a l y s i s of Membrane
Chrambach and D.
Rodbard, S c i e n c e , Acad. S c i . ,
P r o t e i n s , " A. H. Maddy, e d . ,
4.
D.
S u l i t z e a n u and W.
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