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Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
High Resolution Polyacrylamide Gradient Gel Electrophoresis a
Joseph J. Esposito & John F. Obijeski
a
a
Viral Exanthems Branch, Virology Division, Bureau of Laboratories, Center for Disease Control, Public Health Service, U. S. Department of Health, Education, and Welfare , Atlanta, Ga, 30333 Published online: 05 Dec 2006.
To cite this article: Joseph J. Esposito & John F. Obijeski (1976) High Resolution Polyacrylamide Gradient Gel Electrophoresis, Preparative Biochemistry, 6:6, 431-442 To link to this article: http://dx.doi.org/10.1080/00327487608069128
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PREPARATIVE BIOCHPIISTRY , 6 ( 6 ) ,
431-442
(1976)
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H I G H RESOLUTION POLYACRYLAMIDE GRADIENT GEL ELECTROPHORESIS
Joseph J. E s p o s i t o and John F. O b i j e s k i Viral Exanthems Branch, Virology D i v i s i o n , Bureau of L a b o r a t o r i e s , Center f o r D i s e a s e C o n t r o l , P u b l i c H e a l t h S e r v i c e , U.S. Department of H e a l t h , Education, and Welfare, A t l a n t a , Ga. 30333 ABSTRACT A method was developed f o r h i g h r e s o l u t i o n e l e c t r o p h o r e s i s of
p r o t e i n s i n l i n e a r g r a d i e n t (3 t o 30%) p o l y a c r y l a m i d e g e l r o d s i n a n e u t r a l phosphate b u f f e r c o n t a i n i n g 0.1% sodium d o d e c y l s u l f a t e . Well-defined p r o t e i n zones were observed and improved r e s o l u t i o n was a t t a i n e d e s p e c i a l l y f o r low m o l e c u l a r weight p r o t e i n s i n prep-
a r a t i o n s c o n t a i n i n g a v a r i e t y of p o l y p e p t i d e s , e . g . v i r u s e s that a r e o f t e n s e p a r a t e d by c o n t i n u o u s g e l methods.
Electropherograms
of c o n t i n u o u s (8X)and g r a d i e n t (3 t o 30%) g e l s were made of p u r i fied vesicular stomatitis virus, variola virus, Rickettsia rickett-
a, and
a l p h a and b e t a c h a i n s of hemoglobin i n o r d e r t o d e m o n s t r a t e
t h e r e s o l u t i o n of t h e g r a d i e n t system. INTRODUCTION Polyacrylamide g e l e l e c t r o p h o r e s i s (PACE) o f p r o t e i n s i n a system which c o n t a i n s t h e d e t e r g e n t sodium d o d e c y l s u l f a t e (SDS) p r o v i d e s a r e l i a b l e method f o r m o l e c u l a r weight e s t i m a t i o n . 4,lO
431
>
Copyriehr 1 9 - 6 h) Mdricl 1)ekker Inc All Rights Reserved Neither this work nor a n y part may he reproduLcd or rr.lnimitlrd in an). form or hy a n y means. electronlc or mechanical. including photocopying miLrolilrning. J n d recordlng. or by a n y mformatlon storage and retrieval s y s t e m , uirhoui permissmn i n w l t l n g from the publisher
ESPOSITO AND OBIJESKI
432
I n t h i s system SDS d i s s o c i a t e s and d e n a t u r e s p r o t e i n s and n e g a t e s t h e i r n a t i v e c h a r g e t o p e r m i t t h e i 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 by s i z e a s t h e y m i g r a t e toward t h e anode through t h e p o l y a c r y l a m i d e matrix.
By u t i l i z i n g t h e s i e v i n g p r o p e r t i e s of t h e g e l , M a r g o l i s
and Kenrick5 developed a n e l e c t r o p h o r e s i s system, w i t h o u t SDS, com-
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posed o f a c o n t i n u o u s c o n c e n t r a t i o n g r a d i e n t o f p o l y a c r y l a m i d e . They a c h i e v e d h i g h r e s o l u t i o n of n a t i v e p r o t e i n s a t pH 8 . 3 and e s t a b l i s h e d t h a t t h e high r e s o l v i n g power of t h e polyacrylamide g r a d i e n t g e l e l e c t r o p h o r e s i s (PGGE) system was due t o a p r o g r e s s i v e i n c r e a s e i n r e t a r d a t a t i o n of t h e m i g r a t i n g zones u n t i l t h e p r o t e i n s reached a pore l i m i t i n t h e g e l . R e c e n t l y , a PGGE s l a b g e l method w i t h SDS was d e s c r i b e d 2 which c o n t a i n e d a n a l k a l i n e b u f f e r system..’
The p r e s e n t r e p o r t d e s c r i b e s
a h i g h r e s o l u t i o n method f o r SDS-PGGE i n c y l i n d r i c a l g e l s i n a n e u t r a l phosphate b u f f e r .
The g r a d i e n t g e l method r e p o r t e d h e r e was d e v e l -
oped a f t e r a number o f v i r u s p r e p a r a t i o n s were s t u d i e d i n c o n t i n u o u s n e u t r a l phosphate and i n d i s c o n t i n u o u s - a l k a l i n e
( t r i s ) b u f f e r sys-
t e m ~ . ~ , ’ These s t u d i e s r e v e a l e d that t h e m i g r a t i o n of c e r t a i n prot e i n s was d i f f e r e n t i n each system and i n d i c a t e d t h a t a n a l y s i s by
more t h a n me g e l method may b e n e c e s s a r y t o a d e q u a t e l y r e s o l v e proteins.
F u r t h e r , examination by more t h a n one method might p r o v i d e
knowledge of c h a r a c t e r i s t i c s f o r earmarking c e r t a i n p r o t e i n s . Using t h e SDS-PGGE system d e s c r i b e d h e r e w e a s s a y e d v a r i o u s p r o t e i n s and p u r i f i e d p r e p a r a t i o n s of d i f f e r e n t microorganisms and compared t h e r e s u l t s t o t h o s e o b t a i n e d w i t h t h e s t a n d a r d c o n t i n u o u s SDS-PAGE method i n n e u t r a l phosphate.
433
POLYACRYLAMIDE GEL ELECTROPHORESIS
MATERIALS AND METHODS Reagents.
P u r i f i e d a c r y l a m i d e and N,N'-methylenebisacrylamide
were o b t a i n e d from Bio-Rad Labs, Richmond, C a l .
(Bis)
Ammonium p e r s u l f a t e
(AP) and N , N , N 1 ,N1-tetramethylethylenediamine (TEMED) were from East-
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man Kodak Co., Rochester, N.Y.
Other r e a g e n t s i n c l u d e d s p e c t r o -
g r a d e SDS and g l y c e r o l from BDH Chemical Co., Poole, England, and 2-mercaptoethanol
(2-ME) from Calbiochem, L a J o l l a , Cal.
Polypeptides
used as molecular weight s t a n d a r d s were o b t a i n e d from Mann Research Labs, N . Y .
P u r i f i e d p r e p a r a t i o n s of t h e a l p h a and b e t a c h a i n s of
hemoglobin were g e n e r o u s l y s u p p l i e d by Dr. W. Moo-Penn, Hematology D i v i s i o n , Center f o r Disease C o n t r o l , A t l a n t a , Ga."
Viruses and R i c k e t t s i a .
V e s i c u l a r s t o m a t i t i s v i r u s (VSV) and v a r i o l a
v i r u s from i n f e c t e d c e l l c u l t u r e s were p u r i f i e d by d i f f e r e n t i a l , r a t e z o n a l , and e q u i l i b r i u m c e n t r i f u g a t i o n s . *
'
Rickettsia
7
ickettsii
were p u r i f i e d from i n f e c t e d y o l k sacs by methods a l r e a d y d e s c r i b e d . G e l Preparation.
8
Standard 8% (w/v) g e l s f o r c o n t i n u o u s SDS-PAGE were
p r e p a r e d as d e s c r i b e d p r e v i o u s l y 6 i n 0 . 1 M sodium p h o s p h a t e b u f f e r , pH 7 . 0, and 0.1% SDS.
By modifying a t e c h n i q u e d e s c r i b e d by M a r g o l i s and Kenrick',
we
p r e p a r e d b a t c h e s of 100 l i n e a r 3 t o 30% (w/v) p o l y a c r y l a m i d e g r a d i e n t phosphate-SDS g e l r o d s (acry1amide:Bis = 30:O.a) i n s i d e s i l i c o n i z e d p r e c i s i o n b o r e g l a s s c y l i n d e r s (15 cm x 0.6 cm i n n e r d i a m e t e r ) . The g r a d i e n t maker and t h e g e l forming tower f o r t h i s p r o c e d u r e were obt a i n e d from I s o l a b s , I n c . , Akron, Ohio, b u t t h e P l e x i g l a s t o w a h e i g h t
was l e n g t h e n e d t o accommodate t h e 15 cm c y l i n d e r s .
ESPOSITO AND OBIJESKI
434
Four s o l u t i o n s were prepared f o r c a s t i n g g e l s : e t h a n o l , 5% ( v / v ) i n water; s u c r o s e , 30% (w/w) i n water; and s o l u t i o n s h and B of t h e f o r m u l a t i o n l i s t e d below i n Table I. To cast g e l s , t h e least d e n s e s o l u t i o n , e t h a n o l , w a s d r a i n e d
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i n t o t h e tower b a s e f i r s t , s i n c e e t h a n o l serves as a n o v e r l a y a t t h e upper meniscus of t h e g e l .
Next, s o l u t i o n s A and B were d r a i n e d
s i m u l t a n e o u s l y from t h e i r r e s p e c t i v e chambers of t h e g r a d i e n t maker through a mixing t u b e connected t o t h e b a s e of t h e tower t o push t h e e t h a n o l upwards.
S o l u t i o n s A and B were formulated so t h a t t h e mix-
t u r e that flowed f i r s t from t h e g r a d i e n t mixing t u b e w a s 3% polyacrylamide.
The flow rates of s o l u t i o n s A and B, i.e.
t h e t y p e of
g r a d i e n t formed, was c o n t r o l l e d by i n i t i a l l y a d j u s t i n g t h e geometric d e s i g n of t h e g r a d i e n t maker.
The most dense s o l u t i o n , 30% s u c r o s e ,
was used t o push t h e g r a d i e n t o v e r l a i d w i t h 5% e t h a n o l from t h e b a s e of t h e tower upwards t o f i l l t h e 1 5 cm g l a s s t u b e s i n t h e tower.
TABLE I
Reagents f o r P r e p a r i n g 3 t o 30% G r a d i e n t Gels
Reagents
Solution A
Solution B
Acrylamide N,N'-methylenebisacrylamide 1 M sodium phosphate b u f f e r , pR 7.0 S o d i h dodecyl s u l f a t e , 10% (w/v) G l y c e r o l , 50% ( v / v ) Ammonium p e r s u l f a t e , 1 5 % (w/v) N,N,N',N'-tetramethylethylenediamine D i s t i l l e d H20 t o a f i n a l volume of
75.00 2.00 25.00 2.50 75.00 1.25 0.02 250.00
25.00 2.50 25.00 1.25 0.10 250.00
gm gm
ml ml ml
ml ml ml
ml ml
ml ml
ml ml
435
POLYACRYLAMIDE GEL ELECTROPHORESIS
- 30% g r a d i e n t
A complete
linear 3
v i t y feed.
Once t h e t u b e s were f i l l e d t o a h e i g h t of 1 2 c m w i t h
was g e n e r a t e d i n 20 min by g r a -
p o l y a c r y l a m i d e , t h e i n l e t a t t h e tower b a s e was clamped and t h e grad i e n t b l o c k was allowed t o p o l y m e r i z e f o r a t l e a s t 1 hour.
The
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c y l i n d e r s c o n t a i n i n g t h e g r a d i e n t g e l r o d s were c a r e f u l l y removed from t h e b l o c k of polymerized a c r y l a m i d e , and t h e e x c e s s polya c r y l a m i d e was washed o f f .
Before t h e i r u s e g e l s were s t o r e d a t
room t e m p e r a t u r e i n a s o l u t i o n which c o n t a i n e d 0 . 1 El sodium phosp h a t e b u f f e r , 0.01% SDS, 1 5 % g l y c e r o l , 2 mM Na2 EDTA, and 0.01%
NaN3.
I n p r e l i m i n a r y e x p e r i m e n t s , t h e p o l y a c r y l a m i d e g e l r o d s ex-
panded w i t h i n a s h o r t t i m e d u r i n g s t o r a g e and p r o t r u d e d o u t of t h e tube.
T h i s problem w a s c o r r e c t e d by i n c l u d i n g a 5-15% ( v / v ) g l y c e r -
o l g r a d i e n t i n t h e g e l formula and by a d j u s t i n g t h e s t o r a g e b u f f e r t o 15%g l y c e r o l . P r o t e i n samples f o r e l e c t r o p h o r e s i s were d i s s o c i a t e d a t 1OO'C f o r 3 min i n 0.01 M phosphate b u f f e r which c o n t a i n e d 2.5% SDS, 5 % 2-ME,
1 0 % g l y c e r o l , and 0.005% bromphenol b l u e ( d i s s o c i a t i o n b u f f e r ) .
Twenty t o 100 ug of p r o t e i n was l o a d e d o n t o e a c h g e l .
Electrode
b u f f e r w a s 0 . 1 M sodium phosphate, pH 7.0, i n 0.1% SDS.
Electrophor-
esis i n 3-30% g e l s was performed a t a c o n s t a n t c u r r e n t of 4 mA/gel f o r a t least 24 hr t o p e r m i t p r o t e i n s t o m i g r a t e t o t h e i r p o r e limits. I n s t a n d a r d 8% SDS-PAGE, t h e samples were r u n a t 3 mA/gel f o r 1 6 h r , u n t i l t h e bromphenol b l u e reached t h e end of t h e g e l .
After electro-
p h o r e s i s , g e l r o d s were removed by b r e a k i n g t h e g l a s s c y l i n d e r under a s h e e t of 118 i n c h P l e x i g l a s a s d e s c r i b e d by M a i ~ e l . ~
ESPOSITO AM) OBIJESKI
436
Gels were s t a i n e d f o r p r o t e i n f o r 2-8 h r a t 37'C
i n a fixing-
s t a i n i n g s o l u t i o n which c o n t a i n e d 0.2% Coomassie B r i l l i a n t Blue R-250 i n methano1:water:acetic
a c i d (5:5:1).
Unbound dye was removed by
washing t h e g e l r o d s a t 60°C (5-10 changes) in a d e s t a i n s o l u t i o n
(7.5% a c e t i c a c i d and 5% methanol i n w a t e r ) .
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RESULTS E l e c t r o p h o r e t i c P r o g r e s s Curves.
The e l e c t r o p h o r e s i s t i m e n e c e s s a r y
f o r six p r o t e i n molecular weight s t a n d a r d s t o r e a c h t h e i r p o r e l i m i t s i n 3-30% polyacrylamide g r a d i e n t g e l s is shown i n F i g u r e 1. For t h e s e experiments a m i x t u r e of t h e s t a n d a r d s , which ranged i n molecular weight from 12,000 t o 125,000 d a l t o n s and which c o n t a i n e d
5-10 ug of each s t a n d a r d i n 50 u l of d i s s o c i a t i o n b u f f e r , was preThe p r o t e i n s were e l e c t r o p h o r -
pared and a p p l i e d t o s e p a r a t e g e l s .
e s e d a t a c o n s t a n t c u r r e n t of 4 mA/gel, and d u p l i c a t e g e l r o d s were removed and s t a i n e d a t v a r i o u s times d u r i n g e l e c t r o p h o r e s i s . m i g r a t i o n d i s t a n c e f o r each p r o t e i n w a s measured and p l o t t e d .
The The
p r o t e i n s reached t h e i r g r a d i e n t pore l i m i t s by 24 h r ; l o n g e r e l e c t r o p h o r e s i s times (up t o 48 h r ) d i d . n o t i n c r e a s e t h e i r m i g r a t i o n .
The
g e l photograph a t t h e r i g h t of F i g u r e 1 shows t h e m i g r a t i o n d i s t a n c e f o r each p r o t e i n a f t e r 24 h r o f e l e c t r o p h o r e s i s .
The o t h e r minor
p r o t e i n bands i n t h i s photograph r e p r e s e n t i m p u r i t i e s always p r e s e n t i n commercially a v a i l a b l e s t a n d a r d s . R_esolving Power of G r a d i e n t Gels.
The r e s o l v i n g power of g r a d i e n t
g e l s was e v a l u a t e d i n terms of t h e c l a r i t y , compactness, and t o t a l number of p o l y p e p t i d e bands f o r s t a i n e d g r a d i e n t g e l e l e c t r o p h e r o g r a m s
11(
lo( n
9(
E E 8C
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Y
c 7c 0
-6C U g 5c -t
m 40 --
'
30 20 10 10
20 Time (hrs)
30
24
FIGURE 1
Electrophoretic progress curves for s i x protein standards i n 3-30% g r a d i e n t S D S g e l s which show t h e t i m e n e c e s s a r y f o r t h e s t a n d a r d s t o r e a c h t h e i r p o r e l i m i t a t 4 mA c o n s t a n t c u r r e n t . The m i g r a t i o n d i s t a n c e was measured from Coomassie b l u e s t a i n e d g e l s and p l o t t e d a g a i n s t time f o r c y to c h r om e C , 1 2 , 4 0 0 d a l t o n s ( C y ) ; c h y m o t r y p s i n , 25,000 d a l t o n s (Ch); ov al b u m i n , 45,000 d a l t o n s ( 0 ) ; b o v i n e albumin. 68,000 d a l t o n s ( A ) ; p h o s p h o r y l a s e A , 9 2 , 0 0 0 d a l t o n s ( P I ; and b e t a - g a l a c t o s i d a s e . 125,000 d a l t o n s (GI. X s t a i n e d g e l r l e c t r o p h e r o g r a m a f t e r 2 4 h r of e l e c t r o p h o r e s i s i s shown a t t h e r i g h t . E l e c t r o p h o r e s i s was t o u a r d t h e an o d e (+).
438
ESPOSITO AND O B I J E S K I
compared t o s t a n d a r d co n t i n u o u s 8% g e l s .
In one experiment ( F i g . 2 A ) ,
p u r i f i e d hemoglobin a l p h a c h a i n s (15,126 d a l t o n s ) and b e t a c h a i n s (15.866 d a l t o n s ) were co - el ect r o p h o r es ed by b o t h t h e g r a d i e n t and c o n t i n u o u s methods.
These p r o t e i n s , which d i f f e r i n m ole c ula r w e ight
by less t h a n 1000 d a l t o n s , were c l e a r l y s e p a r a t e d by SDS-PGGE (Fig.
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2A, l e f t photograph).
When t h e same material w a s c o-e le c trophore se d
in c o n t i n u o u s 8% g e l s , o n l y a s i n g l e d i f f u s e band was obse rve d ( r i g h t photograph). Because t h e SDS-PGGE system could s e p a r a t e a l p h a and b e t a hemog l o b i n c h a i n s , w e d eci d ed t o examine t h e r e s o l v i n g power f o r o t h e r ,
more complex p r o t e i n mi x t u r es .
W e an al y zed t h e p r o t e i n c o n s t i t u e n t s
of h i g h l y p u r i f i e d p r e p a r a t i o n s of v e s i c u l a r s t o m a t i t i s v i r u s , New J e r s e y s e r o t y p e (VSV), v a r i o l a virus (Harvey), and
&.
r i c k e t t s i i by
SDS-PGGE and by c o n t i n u o u s SDS-PAGE in 8% g e l s ( F i g s . 2B, 2 C , 23).
Electropherograms f o r 3-30% g e l s by SDS-PGGE a r e shown in t h e l e f t photograph, t h o s e f o r 8% g e l s by SDS-PAGE in t h e r i g h t . For VSV (Fig. ZB), t h e s t a i n e d bands of f o u r of t h e f i v e major v i r i o n s t r u c t u r a l proteins’
(L-160,000 d a l t o n s , G-64,000 d a l t o n s , N-52,000 d a l t o n s ,
and M-24,000 d a l t o n s ) ap p ear v e r y compact and d i s t i n c t when compared t o t h e c o n t i n u o u s 8% g e l .
More i mp o r t an t is t h e r e s o l u t i o n i n t h e
r e g i o n of t h e f i f t h p r o t e i n , NS, in which two p r o t e i n s (42,000 d a l t o n s and 40,000 d a l t o n s ) have been s e p a r a t e d .
The NS r e g i o n is d i f -
f i c u l t t o c l e a r l y d emo n s t r at e w i t h s t a i n e d c o n t i n u o u s g e l s . An a ddit i o n a l p r o t e i n n o t found in 8% g e l s , which m igra te d s l i g h t l y in t h e f r o n t of t h e M p r o t e i n , w a s observed i n t h e SDS-PGGE system.
i
'
b
(+I
B
.
1IIM
k'Ns
.!
mn
U C
C
D
FIGURE 2 . S t a i n e d g e l e l e c t r o p h e r o g r a m s t o r t h e a l p h a and b e t a c h a i n s of hemoglobin (ZA), v e s i c u l a r stomat i t i s virus (ZB), v a r i o l a v i r u s ( Z C ) , and r i c k e t t s i i (ZD). The p r o t e i n s were s e p a r a t e d by SDS-PAGE w i t h t h e c o n t i n u o u s 8% system ( r i g h t g e l i n p a n e l ) and by SDS-PGCE w i t h t h e l i n e a r 3-30% system ( l e t g e l i n p a n e l ) . VSV p r o t e i n s have been d e s i g n a t e d by c o n v e n t i o n a l nomenE l e c t r o p h o r e s i s was toward t h e anode (+). c l a t u r e letters.'
A
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W
w
f5
s
'd
4 40
ESPOSITO AND OBIJESKI The s t a i n e d e l e c t r o p h e r o g r a m s o b t a i n e d f o r v a r i o l a v i r u s and
-R.
r i c k e t t s i i are shown i n F i g u r e s 2C and 20, r e s p e c t i v e l y . Pro-
t e i n s i n t h e low m o l e c u l a r w e i g h t r e g i o n (
Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
High Resolution Polyacrylamide Gradient Gel Electrophoresis a
Joseph J. Esposito & John F. Obijeski
a
a
Viral Exanthems Branch, Virology Division, Bureau of Laboratories, Center for Disease Control, Public Health Service, U. S. Department of Health, Education, and Welfare , Atlanta, Ga, 30333 Published online: 05 Dec 2006.
To cite this article: Joseph J. Esposito & John F. Obijeski (1976) High Resolution Polyacrylamide Gradient Gel Electrophoresis, Preparative Biochemistry, 6:6, 431-442 To link to this article: http://dx.doi.org/10.1080/00327487608069128
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PREPARATIVE BIOCHPIISTRY , 6 ( 6 ) ,
431-442
(1976)
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H I G H RESOLUTION POLYACRYLAMIDE GRADIENT GEL ELECTROPHORESIS
Joseph J. E s p o s i t o and John F. O b i j e s k i Viral Exanthems Branch, Virology D i v i s i o n , Bureau of L a b o r a t o r i e s , Center f o r D i s e a s e C o n t r o l , P u b l i c H e a l t h S e r v i c e , U.S. Department of H e a l t h , Education, and Welfare, A t l a n t a , Ga. 30333 ABSTRACT A method was developed f o r h i g h r e s o l u t i o n e l e c t r o p h o r e s i s of
p r o t e i n s i n l i n e a r g r a d i e n t (3 t o 30%) p o l y a c r y l a m i d e g e l r o d s i n a n e u t r a l phosphate b u f f e r c o n t a i n i n g 0.1% sodium d o d e c y l s u l f a t e . Well-defined p r o t e i n zones were observed and improved r e s o l u t i o n was a t t a i n e d e s p e c i a l l y f o r low m o l e c u l a r weight p r o t e i n s i n prep-
a r a t i o n s c o n t a i n i n g a v a r i e t y of p o l y p e p t i d e s , e . g . v i r u s e s that a r e o f t e n s e p a r a t e d by c o n t i n u o u s g e l methods.
Electropherograms
of c o n t i n u o u s (8X)and g r a d i e n t (3 t o 30%) g e l s were made of p u r i fied vesicular stomatitis virus, variola virus, Rickettsia rickett-
a, and
a l p h a and b e t a c h a i n s of hemoglobin i n o r d e r t o d e m o n s t r a t e
t h e r e s o l u t i o n of t h e g r a d i e n t system. INTRODUCTION Polyacrylamide g e l e l e c t r o p h o r e s i s (PACE) o f p r o t e i n s i n a system which c o n t a i n s t h e d e t e r g e n t sodium d o d e c y l s u l f a t e (SDS) p r o v i d e s a r e l i a b l e method f o r m o l e c u l a r weight e s t i m a t i o n . 4,lO
431
>
Copyriehr 1 9 - 6 h) Mdricl 1)ekker Inc All Rights Reserved Neither this work nor a n y part may he reproduLcd or rr.lnimitlrd in an). form or hy a n y means. electronlc or mechanical. including photocopying miLrolilrning. J n d recordlng. or by a n y mformatlon storage and retrieval s y s t e m , uirhoui permissmn i n w l t l n g from the publisher
ESPOSITO AND OBIJESKI
432
I n t h i s system SDS d i s s o c i a t e s and d e n a t u r e s p r o t e i n s and n e g a t e s t h e i r n a t i v e c h a r g e t o p e r m i t t h e i 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 by s i z e a s t h e y m i g r a t e toward t h e anode through t h e p o l y a c r y l a m i d e matrix.
By u t i l i z i n g t h e s i e v i n g p r o p e r t i e s of t h e g e l , M a r g o l i s
and Kenrick5 developed a n e l e c t r o p h o r e s i s system, w i t h o u t SDS, com-
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posed o f a c o n t i n u o u s c o n c e n t r a t i o n g r a d i e n t o f p o l y a c r y l a m i d e . They a c h i e v e d h i g h r e s o l u t i o n of n a t i v e p r o t e i n s a t pH 8 . 3 and e s t a b l i s h e d t h a t t h e high r e s o l v i n g power of t h e polyacrylamide g r a d i e n t g e l e l e c t r o p h o r e s i s (PGGE) system was due t o a p r o g r e s s i v e i n c r e a s e i n r e t a r d a t a t i o n of t h e m i g r a t i n g zones u n t i l t h e p r o t e i n s reached a pore l i m i t i n t h e g e l . R e c e n t l y , a PGGE s l a b g e l method w i t h SDS was d e s c r i b e d 2 which c o n t a i n e d a n a l k a l i n e b u f f e r system..’
The p r e s e n t r e p o r t d e s c r i b e s
a h i g h r e s o l u t i o n method f o r SDS-PGGE i n c y l i n d r i c a l g e l s i n a n e u t r a l phosphate b u f f e r .
The g r a d i e n t g e l method r e p o r t e d h e r e was d e v e l -
oped a f t e r a number o f v i r u s p r e p a r a t i o n s were s t u d i e d i n c o n t i n u o u s n e u t r a l phosphate and i n d i s c o n t i n u o u s - a l k a l i n e
( t r i s ) b u f f e r sys-
t e m ~ . ~ , ’ These s t u d i e s r e v e a l e d that t h e m i g r a t i o n of c e r t a i n prot e i n s was d i f f e r e n t i n each system and i n d i c a t e d t h a t a n a l y s i s by
more t h a n me g e l method may b e n e c e s s a r y t o a d e q u a t e l y r e s o l v e proteins.
F u r t h e r , examination by more t h a n one method might p r o v i d e
knowledge of c h a r a c t e r i s t i c s f o r earmarking c e r t a i n p r o t e i n s . Using t h e SDS-PGGE system d e s c r i b e d h e r e w e a s s a y e d v a r i o u s p r o t e i n s and p u r i f i e d p r e p a r a t i o n s of d i f f e r e n t microorganisms and compared t h e r e s u l t s t o t h o s e o b t a i n e d w i t h t h e s t a n d a r d c o n t i n u o u s SDS-PAGE method i n n e u t r a l phosphate.
433
POLYACRYLAMIDE GEL ELECTROPHORESIS
MATERIALS AND METHODS Reagents.
P u r i f i e d a c r y l a m i d e and N,N'-methylenebisacrylamide
were o b t a i n e d from Bio-Rad Labs, Richmond, C a l .
(Bis)
Ammonium p e r s u l f a t e
(AP) and N , N , N 1 ,N1-tetramethylethylenediamine (TEMED) were from East-
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man Kodak Co., Rochester, N.Y.
Other r e a g e n t s i n c l u d e d s p e c t r o -
g r a d e SDS and g l y c e r o l from BDH Chemical Co., Poole, England, and 2-mercaptoethanol
(2-ME) from Calbiochem, L a J o l l a , Cal.
Polypeptides
used as molecular weight s t a n d a r d s were o b t a i n e d from Mann Research Labs, N . Y .
P u r i f i e d p r e p a r a t i o n s of t h e a l p h a and b e t a c h a i n s of
hemoglobin were g e n e r o u s l y s u p p l i e d by Dr. W. Moo-Penn, Hematology D i v i s i o n , Center f o r Disease C o n t r o l , A t l a n t a , Ga."
Viruses and R i c k e t t s i a .
V e s i c u l a r s t o m a t i t i s v i r u s (VSV) and v a r i o l a
v i r u s from i n f e c t e d c e l l c u l t u r e s were p u r i f i e d by d i f f e r e n t i a l , r a t e z o n a l , and e q u i l i b r i u m c e n t r i f u g a t i o n s . *
'
Rickettsia
7
ickettsii
were p u r i f i e d from i n f e c t e d y o l k sacs by methods a l r e a d y d e s c r i b e d . G e l Preparation.
8
Standard 8% (w/v) g e l s f o r c o n t i n u o u s SDS-PAGE were
p r e p a r e d as d e s c r i b e d p r e v i o u s l y 6 i n 0 . 1 M sodium p h o s p h a t e b u f f e r , pH 7 . 0, and 0.1% SDS.
By modifying a t e c h n i q u e d e s c r i b e d by M a r g o l i s and Kenrick',
we
p r e p a r e d b a t c h e s of 100 l i n e a r 3 t o 30% (w/v) p o l y a c r y l a m i d e g r a d i e n t phosphate-SDS g e l r o d s (acry1amide:Bis = 30:O.a) i n s i d e s i l i c o n i z e d p r e c i s i o n b o r e g l a s s c y l i n d e r s (15 cm x 0.6 cm i n n e r d i a m e t e r ) . The g r a d i e n t maker and t h e g e l forming tower f o r t h i s p r o c e d u r e were obt a i n e d from I s o l a b s , I n c . , Akron, Ohio, b u t t h e P l e x i g l a s t o w a h e i g h t
was l e n g t h e n e d t o accommodate t h e 15 cm c y l i n d e r s .
ESPOSITO AND OBIJESKI
434
Four s o l u t i o n s were prepared f o r c a s t i n g g e l s : e t h a n o l , 5% ( v / v ) i n water; s u c r o s e , 30% (w/w) i n water; and s o l u t i o n s h and B of t h e f o r m u l a t i o n l i s t e d below i n Table I. To cast g e l s , t h e least d e n s e s o l u t i o n , e t h a n o l , w a s d r a i n e d
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i n t o t h e tower b a s e f i r s t , s i n c e e t h a n o l serves as a n o v e r l a y a t t h e upper meniscus of t h e g e l .
Next, s o l u t i o n s A and B were d r a i n e d
s i m u l t a n e o u s l y from t h e i r r e s p e c t i v e chambers of t h e g r a d i e n t maker through a mixing t u b e connected t o t h e b a s e of t h e tower t o push t h e e t h a n o l upwards.
S o l u t i o n s A and B were formulated so t h a t t h e mix-
t u r e that flowed f i r s t from t h e g r a d i e n t mixing t u b e w a s 3% polyacrylamide.
The flow rates of s o l u t i o n s A and B, i.e.
t h e t y p e of
g r a d i e n t formed, was c o n t r o l l e d by i n i t i a l l y a d j u s t i n g t h e geometric d e s i g n of t h e g r a d i e n t maker.
The most dense s o l u t i o n , 30% s u c r o s e ,
was used t o push t h e g r a d i e n t o v e r l a i d w i t h 5% e t h a n o l from t h e b a s e of t h e tower upwards t o f i l l t h e 1 5 cm g l a s s t u b e s i n t h e tower.
TABLE I
Reagents f o r P r e p a r i n g 3 t o 30% G r a d i e n t Gels
Reagents
Solution A
Solution B
Acrylamide N,N'-methylenebisacrylamide 1 M sodium phosphate b u f f e r , pR 7.0 S o d i h dodecyl s u l f a t e , 10% (w/v) G l y c e r o l , 50% ( v / v ) Ammonium p e r s u l f a t e , 1 5 % (w/v) N,N,N',N'-tetramethylethylenediamine D i s t i l l e d H20 t o a f i n a l volume of
75.00 2.00 25.00 2.50 75.00 1.25 0.02 250.00
25.00 2.50 25.00 1.25 0.10 250.00
gm gm
ml ml ml
ml ml ml
ml ml
ml ml
ml ml
435
POLYACRYLAMIDE GEL ELECTROPHORESIS
- 30% g r a d i e n t
A complete
linear 3
v i t y feed.
Once t h e t u b e s were f i l l e d t o a h e i g h t of 1 2 c m w i t h
was g e n e r a t e d i n 20 min by g r a -
p o l y a c r y l a m i d e , t h e i n l e t a t t h e tower b a s e was clamped and t h e grad i e n t b l o c k was allowed t o p o l y m e r i z e f o r a t l e a s t 1 hour.
The
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c y l i n d e r s c o n t a i n i n g t h e g r a d i e n t g e l r o d s were c a r e f u l l y removed from t h e b l o c k of polymerized a c r y l a m i d e , and t h e e x c e s s polya c r y l a m i d e was washed o f f .
Before t h e i r u s e g e l s were s t o r e d a t
room t e m p e r a t u r e i n a s o l u t i o n which c o n t a i n e d 0 . 1 El sodium phosp h a t e b u f f e r , 0.01% SDS, 1 5 % g l y c e r o l , 2 mM Na2 EDTA, and 0.01%
NaN3.
I n p r e l i m i n a r y e x p e r i m e n t s , t h e p o l y a c r y l a m i d e g e l r o d s ex-
panded w i t h i n a s h o r t t i m e d u r i n g s t o r a g e and p r o t r u d e d o u t of t h e tube.
T h i s problem w a s c o r r e c t e d by i n c l u d i n g a 5-15% ( v / v ) g l y c e r -
o l g r a d i e n t i n t h e g e l formula and by a d j u s t i n g t h e s t o r a g e b u f f e r t o 15%g l y c e r o l . P r o t e i n samples f o r e l e c t r o p h o r e s i s were d i s s o c i a t e d a t 1OO'C f o r 3 min i n 0.01 M phosphate b u f f e r which c o n t a i n e d 2.5% SDS, 5 % 2-ME,
1 0 % g l y c e r o l , and 0.005% bromphenol b l u e ( d i s s o c i a t i o n b u f f e r ) .
Twenty t o 100 ug of p r o t e i n was l o a d e d o n t o e a c h g e l .
Electrode
b u f f e r w a s 0 . 1 M sodium phosphate, pH 7.0, i n 0.1% SDS.
Electrophor-
esis i n 3-30% g e l s was performed a t a c o n s t a n t c u r r e n t of 4 mA/gel f o r a t least 24 hr t o p e r m i t p r o t e i n s t o m i g r a t e t o t h e i r p o r e limits. I n s t a n d a r d 8% SDS-PAGE, t h e samples were r u n a t 3 mA/gel f o r 1 6 h r , u n t i l t h e bromphenol b l u e reached t h e end of t h e g e l .
After electro-
p h o r e s i s , g e l r o d s were removed by b r e a k i n g t h e g l a s s c y l i n d e r under a s h e e t of 118 i n c h P l e x i g l a s a s d e s c r i b e d by M a i ~ e l . ~
ESPOSITO AM) OBIJESKI
436
Gels were s t a i n e d f o r p r o t e i n f o r 2-8 h r a t 37'C
i n a fixing-
s t a i n i n g s o l u t i o n which c o n t a i n e d 0.2% Coomassie B r i l l i a n t Blue R-250 i n methano1:water:acetic
a c i d (5:5:1).
Unbound dye was removed by
washing t h e g e l r o d s a t 60°C (5-10 changes) in a d e s t a i n s o l u t i o n
(7.5% a c e t i c a c i d and 5% methanol i n w a t e r ) .
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RESULTS E l e c t r o p h o r e t i c P r o g r e s s Curves.
The e l e c t r o p h o r e s i s t i m e n e c e s s a r y
f o r six p r o t e i n molecular weight s t a n d a r d s t o r e a c h t h e i r p o r e l i m i t s i n 3-30% polyacrylamide g r a d i e n t g e l s is shown i n F i g u r e 1. For t h e s e experiments a m i x t u r e of t h e s t a n d a r d s , which ranged i n molecular weight from 12,000 t o 125,000 d a l t o n s and which c o n t a i n e d
5-10 ug of each s t a n d a r d i n 50 u l of d i s s o c i a t i o n b u f f e r , was preThe p r o t e i n s were e l e c t r o p h o r -
pared and a p p l i e d t o s e p a r a t e g e l s .
e s e d a t a c o n s t a n t c u r r e n t of 4 mA/gel, and d u p l i c a t e g e l r o d s were removed and s t a i n e d a t v a r i o u s times d u r i n g e l e c t r o p h o r e s i s . m i g r a t i o n d i s t a n c e f o r each p r o t e i n w a s measured and p l o t t e d .
The The
p r o t e i n s reached t h e i r g r a d i e n t pore l i m i t s by 24 h r ; l o n g e r e l e c t r o p h o r e s i s times (up t o 48 h r ) d i d . n o t i n c r e a s e t h e i r m i g r a t i o n .
The
g e l photograph a t t h e r i g h t of F i g u r e 1 shows t h e m i g r a t i o n d i s t a n c e f o r each p r o t e i n a f t e r 24 h r o f e l e c t r o p h o r e s i s .
The o t h e r minor
p r o t e i n bands i n t h i s photograph r e p r e s e n t i m p u r i t i e s always p r e s e n t i n commercially a v a i l a b l e s t a n d a r d s . R_esolving Power of G r a d i e n t Gels.
The r e s o l v i n g power of g r a d i e n t
g e l s was e v a l u a t e d i n terms of t h e c l a r i t y , compactness, and t o t a l number of p o l y p e p t i d e bands f o r s t a i n e d g r a d i e n t g e l e l e c t r o p h e r o g r a m s
11(
lo( n
9(
E E 8C
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Y
c 7c 0
-6C U g 5c -t
m 40 --
'
30 20 10 10
20 Time (hrs)
30
24
FIGURE 1
Electrophoretic progress curves for s i x protein standards i n 3-30% g r a d i e n t S D S g e l s which show t h e t i m e n e c e s s a r y f o r t h e s t a n d a r d s t o r e a c h t h e i r p o r e l i m i t a t 4 mA c o n s t a n t c u r r e n t . The m i g r a t i o n d i s t a n c e was measured from Coomassie b l u e s t a i n e d g e l s and p l o t t e d a g a i n s t time f o r c y to c h r om e C , 1 2 , 4 0 0 d a l t o n s ( C y ) ; c h y m o t r y p s i n , 25,000 d a l t o n s (Ch); ov al b u m i n , 45,000 d a l t o n s ( 0 ) ; b o v i n e albumin. 68,000 d a l t o n s ( A ) ; p h o s p h o r y l a s e A , 9 2 , 0 0 0 d a l t o n s ( P I ; and b e t a - g a l a c t o s i d a s e . 125,000 d a l t o n s (GI. X s t a i n e d g e l r l e c t r o p h e r o g r a m a f t e r 2 4 h r of e l e c t r o p h o r e s i s i s shown a t t h e r i g h t . E l e c t r o p h o r e s i s was t o u a r d t h e an o d e (+).
438
ESPOSITO AND O B I J E S K I
compared t o s t a n d a r d co n t i n u o u s 8% g e l s .
In one experiment ( F i g . 2 A ) ,
p u r i f i e d hemoglobin a l p h a c h a i n s (15,126 d a l t o n s ) and b e t a c h a i n s (15.866 d a l t o n s ) were co - el ect r o p h o r es ed by b o t h t h e g r a d i e n t and c o n t i n u o u s methods.
These p r o t e i n s , which d i f f e r i n m ole c ula r w e ight
by less t h a n 1000 d a l t o n s , were c l e a r l y s e p a r a t e d by SDS-PGGE (Fig.
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2A, l e f t photograph).
When t h e same material w a s c o-e le c trophore se d
in c o n t i n u o u s 8% g e l s , o n l y a s i n g l e d i f f u s e band was obse rve d ( r i g h t photograph). Because t h e SDS-PGGE system could s e p a r a t e a l p h a and b e t a hemog l o b i n c h a i n s , w e d eci d ed t o examine t h e r e s o l v i n g power f o r o t h e r ,
more complex p r o t e i n mi x t u r es .
W e an al y zed t h e p r o t e i n c o n s t i t u e n t s
of h i g h l y p u r i f i e d p r e p a r a t i o n s of v e s i c u l a r s t o m a t i t i s v i r u s , New J e r s e y s e r o t y p e (VSV), v a r i o l a virus (Harvey), and
&.
r i c k e t t s i i by
SDS-PGGE and by c o n t i n u o u s SDS-PAGE in 8% g e l s ( F i g s . 2B, 2 C , 23).
Electropherograms f o r 3-30% g e l s by SDS-PGGE a r e shown in t h e l e f t photograph, t h o s e f o r 8% g e l s by SDS-PAGE in t h e r i g h t . For VSV (Fig. ZB), t h e s t a i n e d bands of f o u r of t h e f i v e major v i r i o n s t r u c t u r a l proteins’
(L-160,000 d a l t o n s , G-64,000 d a l t o n s , N-52,000 d a l t o n s ,
and M-24,000 d a l t o n s ) ap p ear v e r y compact and d i s t i n c t when compared t o t h e c o n t i n u o u s 8% g e l .
More i mp o r t an t is t h e r e s o l u t i o n i n t h e
r e g i o n of t h e f i f t h p r o t e i n , NS, in which two p r o t e i n s (42,000 d a l t o n s and 40,000 d a l t o n s ) have been s e p a r a t e d .
The NS r e g i o n is d i f -
f i c u l t t o c l e a r l y d emo n s t r at e w i t h s t a i n e d c o n t i n u o u s g e l s . An a ddit i o n a l p r o t e i n n o t found in 8% g e l s , which m igra te d s l i g h t l y in t h e f r o n t of t h e M p r o t e i n , w a s observed i n t h e SDS-PGGE system.
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FIGURE 2 . S t a i n e d g e l e l e c t r o p h e r o g r a m s t o r t h e a l p h a and b e t a c h a i n s of hemoglobin (ZA), v e s i c u l a r stomat i t i s virus (ZB), v a r i o l a v i r u s ( Z C ) , and r i c k e t t s i i (ZD). The p r o t e i n s were s e p a r a t e d by SDS-PAGE w i t h t h e c o n t i n u o u s 8% system ( r i g h t g e l i n p a n e l ) and by SDS-PGCE w i t h t h e l i n e a r 3-30% system ( l e t g e l i n p a n e l ) . VSV p r o t e i n s have been d e s i g n a t e d by c o n v e n t i o n a l nomenE l e c t r o p h o r e s i s was toward t h e anode (+). c l a t u r e letters.'
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ESPOSITO AND OBIJESKI The s t a i n e d e l e c t r o p h e r o g r a m s o b t a i n e d f o r v a r i o l a v i r u s and
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r i c k e t t s i i are shown i n F i g u r e s 2C and 20, r e s p e c t i v e l y . Pro-
t e i n s i n t h e low m o l e c u l a r w e i g h t r e g i o n (
