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Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
An Improved System for Preparative Starch Block Electrophoresis a
Philip C. Kelleher & Carol J. Smith
a
a
Department of Medicine , University of Vermont College of Medicine Burlington , Vermont, 05401, U.S.A. Published online: 05 Dec 2006.
To cite this article: Philip C. Kelleher & Carol J. Smith (1975) An Improved System for Preparative Starch Block Electrophoresis, Preparative Biochemistry, 5:5-6, 413-422, DOI: 10.1080/00327487508061588 To link to this article: http://dx.doi.org/10.1080/00327487508061588
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [UQ Library] at 02:01 20 November 2014
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-andconditions
PREPARATIVE BIOCHEMISTRY, 5(5 & 6), 413-422 (1975)
AN IMPROVED SYSTEM FOR PREPARATIVE STARCH BLOCK ELECTROPHORESIS
P h i l i p C. Kelleher and Carol J. Smith Department o f Medicine, U n i v e r s i t y o f Vermont College o f Medicine Burlington, Vermont, 05401 U.S.A.
Downloaded by [UQ Library] at 02:01 20 November 2014
ABSTRACT
A s t a r c h block e l e c t r o p h o r e s i s system i s described which i n cludes ( a ) a s i n g l e - u n i t L u c i t e e l e c t r o p h o r e s i s chamber, ( b ) temperature c o n t r o l w i t h a c i r c u l a t i n g water bath and c a s t i n g r e s i n coated brass c o o l i n g p l a t e , ( c ) continuous m o n i t o r i n g o f b l o c k surface temperature and ( d ) an easi ly-assembled apparatus f o r r a p i d e l u t i o n o f p r o t e i n from t h e s t a r c h segments. INTRODUCTION Starch block electrophoresis has been used w i d e l y f o r prep a r a t i v e - s c a l e separation o f proteins, b u t t h e apparatus used i s o f t e n make-shift, cumbersome and i n f l e x i b l e , w i t h inadequate cooling.
This r e p o r t describes several m o d i f i c a t i o n s o f t h e o r i g i n a l
method o f Kunkel and S l a t e r 1 which i n c l u d e an enclosed s i n g l e - u n i t electrophoresis chamber, c o n t r o l l e d and constant c o o l i n g o f t h e block w i t h continuous surface temperature monitoring, and a u n i f i e d apparatus f o r r a p i d simultaneous e l u t i o n o f t h e p r o t e i n from the s t a r c h segments.
413 Copyright 0 1976 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 information storage and retrieval system, without permission in writing from the publisher.
KELLEHER AND SMITH
4 14 APPARATUS AND PROCEDURES
F i g . 1 shows a s c a l e drawing o f t h e L u c i t e e l e c t r o p h o r e s i s chamber c o n s t r u c t e d by t h e U n i v e r s i t y o f Vermont I n s t r u m e n t a t i o n and Model F a c i l i t y . 1 of
The two smaller, o u t e r r e s e r v o i r s h o l d 3.5
0.0511 b a r b i t a l b u f f e r , pH 8.6; t h e two l a r g e r , i n n e r r e s e r -
Downloaded by [UQ Library] at 02:01 20 November 2014
v o i r s , 7.0 1 o f 0.2M sodium phosphate b u f f e r , pH 7.5. tubes o f 30mn O.D.
Four U-
g l a s s t u b i n g , w i t h sidearms t o a l l o w f i l l i n g
b y s u c t i o n , serve as s a l t b r i d g e s between t h e b a r b i t a l and phosphate b u f f e r r e s e r v o i r s a t each end o f t h e chamber.
A short
l e n g t h o f rubber t u b i n g i s p l a c e d between t h e phosphate b u f f e r reservoirs t o equalize the b u f f e r levels.
Platinum wire elec-
t r o d e s r u n t h e e n t i r e l e n g t h o f t h e bottoms o f t h e two i n n e r chambers and connect t o two male banana p l u g s molded i n t o one end o f t h e chamber.
The power s u p p l y i s connected t o two
female banana p l u g s which a r e molded i n t o t h e l i d o f t h e e l e c t r o p h o r e s i s chamber, t h u s p r o v i d i n g an i n t e r l o c k system f o r t h e apparatus.
I n t h i s l a b o r a t o r y t h e e l e c t r o p h o r e s i s chamber and
t h e c o o l i n g p l a t e d e s c r i b e d below a r e always k e p t i n a c o n s t a n t temperature room a t 4°C. C o n t r o l l e d c o o l i n g o f t h e b l o c k i s p r o v i d e d by a brass p l a t e which was c o n s t r u c t e d by t h e U n i v e r s i t y o f Vermont Instrumentat i o n and Model F a c i l i t y . 6.4mm O.D.
For t h e c i r c u l a t i o n o f c o o l a n t f l u i d ,
copper t u b i n g was welded t o t h e undersurface o f t h e
p l a t e i n a continuous " S " p a t t e r n .
I n order t o provide a cool-
i n g p l a t e w i t h h i g h thermal c o n d u c t i v i t y , y e t low e l e c t r i c a l con-
415
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STARCH BLOCK ELECTROPHORESIS
/
FIGURE 1 Scale drawing o f t h e L u c i t e e l e c t r o p h o r e s i s chamber. The g l a s s p l a t e and s t a r c h b l o c k , which r e s t on t o p o f t h e c o o l i n g p l a t e d u r i n g o p e r a t i o n , have been o m i t t e d f r o m t h e drawing f o r t h e sake o f s i m p l i c i t y . Dimensions a r e given i n centimeters.
d u c t i v i t y , t h e brass p l a t e was covered w i t h a c a s t i n g r e s i n ( S t y c a s t No. 2850-KT,
Emerson and Cuming, Inc., Canton, Mass.)
t o f i n a l dimensions o f 63.5cm x 50.2cm x 1.9cm ( w x 1 x t ) . perties o f the r e s i n include:
2 FT /"F/2.54cm;
thermal c o n d u c t i v i t y , 30 BTU/hr/
d i e l e c t r i c constant, 410 V/25.4pm;
t i v i t y , 1015 ohms-cm.
Pro-
volume r e s i s -
The brass p l a t e was p l a c e d i n a mold, and
KELLEHER AND SMITH
416
the liquid resin was poured i n t o form a layer around the plate. The resin was allowed t o harden, and any i r r e g u l a r i t i e s in the surface were corrected by f i l l i n g and hand-lapping.
The plate
res t s on the Lucite buffer reservoir dividers, and the coolant in l e t and o u t l e t tubes extend t h r o u g h grooves cut in one end of the electrophoresis chamber. Water i s circulated through the Downloaded by [UQ Library] at 02:01 20 November 2014
cooling plate by means of a Forma-Temp Model 2220 bath and c i r culator (Forma Sc ie nti fi c , Marietta, Ohio). equi 1i brated a t a temperature of 2°C.
The system i s
After the electrophoretic
separation i s begun, the b a t h temperature i s lowered t o -3°C which maintains the temperature of the upper surface of the block a t 11°C. Block surface temperature i s monitored with a YSI model 42SC telethermometer (Fisher Sc ie nti fi c Co.) with a 4.8mn-diameter surface temperature probe taped t o the polyethylene film covering the block's upper surface. The starch slab i s supported by a 0.64cm-thickY 50.8cmlong and 33.0cm- or 6l.Ocm-wide glass plate framed by Lucite blocks (University of Vermont Instrumentation and Model Facil i t y ) o f various lengths, 5.lcm wide and 2.4cm thick.
The
two Lucite blocks framing the length of the starch slab are 53.3cm long while the other two are 33.0cm o r 61.0cm long, according t o the width of the glass plate.
A sheet of 51um-
thick polyethylene film (Commercial Plastics and Supply Corp., New York, N . Y . )
the same length (50.8cm) as and 30.5cm wider
than the glass plate i s centered on t he moist glass plate.
STARCH BLOCK ELECTROPHORESIS
417
F i l t e r paper wicks are placed along the two widths o f t h e glass p l a t e t o a i d i n t h e d r y i n g o f t h e s t a r c h s l u r r y .
The
t h i c k s t a r c h s l u r r y i s poured i n t o t h i s mold t o a thickness o f 1.25cm and p a r t i a l l y d r i e d w i t h an e l e c t r c fan j u s t u n t i l the s t a r c h slab maintains i t s shape when the L u c i t e frame i s removed.
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A narrow trough i s c u t i n t h e starch w i t h a spatula i n a l i n e perpendicular t o t h e d i r e c t i o n o f m i g r a t i o n and a t a p o i n t between 5.lcm and 17.8cm from t h e cathode end, depending upon t h e expected e l e c t r o p h o r e t i c m i g r a t i o n o f t h e p r o t e i n s .
The
trough i s f i l l e d w i t h the p r o t e i n sample, approximately 0.4 m l / cm o f trough length.
The concentration o f t h e p r o t e i n s o l u t i o n
may be as high as 60 mg/ml, f o r example, whole serum, b u t t h e concentration o f any group o f p r o t e i n s w i t h s i m i l a r e l e c t r o p h o r e t i c m o b i l i t i e s should n o t exceed 30-35 mg/ml f o r optimal resolution.
For separations o f serum proteins, marker samples
o f homologous albumin pre-stained w i t h bromphenol blue may be placed a t t h e o r i g i n , 2.5401 i n from each edge.
A second
sheet o f polyethylene f i l m 48.3cm l o n g i s centered and smoothed over t h e surface o f t h e starch slab.
The s t a r c h slab and glass
p l a t e a r e placed on the c o o l i n g p l a t e and t h e exposed s t a r c h ends covered w i t h b a r b i t a l buffer-soaked c o t t o n t o w e l i n g wicks which extend i n t o t h e b a r b i t a l b u f f e r r e s e r v o i r s .
The wicks a r e
covered w i t h a d d i t i o n a l polyethylene f i l m t o prevent drying, and t h e e n t i r e s t a r c h slab i s wrapped f i r m l y w i t h t h e excess widths o f polyethylene f i l m .
The system i s allowed t o e q u i l i -
KELLEHER AND SMITH
418
brate f o r one-half hour in the electrophoresis chamber with cooling.
The electrophoretic separation i s performed a t a
power supply s et t ing of 600 V, yielding an electr ical f i e l d strength of 8 V/cm, f o r approximately 38 hours or 26 hours for origins a t 5.lcm or 17.8cm respectively from the cathode end. The current i s 90 mA when using the 33.0cm-wide block and 140 mA Downloaded by [UQ Library] at 02:01 20 November 2014
when using the 61.0cm-wide block.
Upon completion of the r u n ,
the starch block i s dried a t room temperature until a small piece maintains i t s shape when held on a spatula.
Forty 1.25cm
sli c es are cut with a spatula, perpendicular t o the direction of electrophoretic migration. Fig. 2 shows the apparatus used t o e lute the proteins from the starch segments.
Each unit consists of a 7.2cm diameter
polypropylene aBuchler funnel (Fisher Sc ie ntif ic Co. ) covered with a 7.0cm Whatman No. 1 f i l t e r paper c i r c l e .
The funnel i s
inserted t h r o u g h a No. 5 1/2 rubber stopper into a 2.7cm I.D. x 17.7cm polypropylene sidearm t e s t tube (Bel-Art Products, Pequannock, N.J.).
The units s i t in a wooden support with
holes of 3.0cm diameter d r i l l e d a t 8.5cm intervals, and a r e interconnected i n se ri e s in an elongated "U" by means of 6.4mm polypropylene T-shaped connecting tubes and 6.4m I.D. vacuum tubing.
The c i r c u i t i s completed by a Y-shaped connecting tube
t o a water vacuum f i l t e r pump. Starch segments a re packed firmly into each funnel with the f l a t end of the plunger from a 50-1111 polyethylene syringe.
A
volume of 0.15M sodium chloride o r suitable buffer a t least 50%
STARCH BLOCK ELECTROPHORESIS
419
c Q)
-0
0
0
3 Q)
r c,
c .r
c
0
.r
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c, .r
v)
0
n c
.r
L Q)
c r
V 3
m
r
c,
.r
3 v)
S
c,
m L m
n n m
c
0 .r
c, 3
4 20
WLLEHER AND SMITH
greater than the i n i t i a l sample volume i s layered immediately over each packed s t a r c h segment.
When a l l funnels are packed,
vacuum i s a p p l i e d t o t h e tubes and t h e e l u t i o n i s completed w i t h i n seconds. RESULTS AND DISCUSSION Fig. 3 demonstrates t h e e l e c t r o p h o r e t i c separation o f ( a )
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t h e p r o t e i n s present i n a d u l t r a t serum and (b) a preparation o f a d u l t r a t serum macroglobulins.
The packed and e l u t e d s t a r c h
segments were r e - e l u t e d w i t h a second a l i q u o t o f b u f f e r ; no f u r t h e r e l u t i o n o f p r o t e i n demonstrated t h e completeness o f sample removal by t h i s e l u t i o n system.
Recovery o f serum pro-
t e i n s which have been applied t o t h e s t a r c h block i s 100%. Preparative separations o f l a r g e amounts o f complex mixt u r e s o f p r o t e i n s such as these can be achieved more e f f i c i e n t l y by starch block electrophoresis than by column chromatography using an ion-exchange r e s i n such as DEAE-Sephadex.
I n addition,
t h i s s t a r c h block electrophoresis apparatus o f f e r s many o t h e r advantages:
easy assembly and storage, c o n t r o l and m o n i t o r i n g
o f block temperature, f l e x i b i l i t y i n b l o c k s i z e and number o f samples, and r a p i d e l u t i o n of t h e p r o t e i n samples.
The e l e c t r o -
phoresis chamber i s safe f o r t h e operator and f o r personnel worki n g i n the v i c i n i t y since i t i s an enclosed system.
Circulation
o f r e f r i g e r a t e d coolant i s e s s e n t i a l f o r maintaining block temperatures below 15°C; w i t h o u t c i r c u l a t i o n , block surface temperatures may reach 30-35°C.
This p r e p a r a t i v e system i s a l s o
s u i t a b l e f o r use w i t h Geon o r Pevikon as t h e supporting medium.
STARCH BLOCK ELECTROPHORESIS
421
1.50(
1.ooc
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.500 c, h .r
v)
c
Q,
0
U
F
rc)
V .r
c,
n O
1.
1.
f r a c t i o n number FIGURE 3 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 o f a d u l t r a t serum p r o t e i n s (above) and a d u l t r a t serum m a c r o g l o b u l i n s (below). Arrows on t h e v e r t i c a l axes denote t h e p o s i t i o n s o f t h e electrophoretic origins.
Compared t o s e r i a l single-sample e l u t i o n procedures, t h i s s i m u l taneous e l u t i o n system i s t w i c e as f a s t when performed by t h e same l a b o r a t o r y personnel.
422
KELLEHER AND SMITH
ACKNOWLEDGMENTS The design and t e s t i n g o f t h i s experimental system has been supported by t h e f o l l o w i n g g r a n t s : U.S.P.H.S.
U.S.P.H.S.
Grant GM 11547;
Grant HD 03483; Vermont H e a r t A s s o c i a t i o n Grant AG 63;
American Cancer Society, Vermont D i v i s i o n , Grant #5; U n i v e r s i t y o f Vermont General Research Support Fund; Damon Runyon Memorial
Downloaded by [UQ Library] at 02:01 20 November 2014
Fund f o r Cancer Research Grant DRG-1184.
We wish t o thank Mr.
George L u h r and t h e s t a f f o f t h e U n i v e r s i t y o f Vermont I n s t r u mentation and Model F a c i l i t y f o r s e l e c t i o n o f t h e c a s t i n g r e s i n used i n f a b r i c a t i n g t h e c o o l i n g p l a t e and f o r c o n s t r u c t i o n o f t h e apparatus. REFERENCES
1.
H.G.
Kunkei and R.J. S l a t e r , Proc. SOC. E x p t l . B i o l . Med.,
80, 42-44 (1952).
Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
An Improved System for Preparative Starch Block Electrophoresis a
Philip C. Kelleher & Carol J. Smith
a
a
Department of Medicine , University of Vermont College of Medicine Burlington , Vermont, 05401, U.S.A. Published online: 05 Dec 2006.
To cite this article: Philip C. Kelleher & Carol J. Smith (1975) An Improved System for Preparative Starch Block Electrophoresis, Preparative Biochemistry, 5:5-6, 413-422, DOI: 10.1080/00327487508061588 To link to this article: http://dx.doi.org/10.1080/00327487508061588
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [UQ Library] at 02:01 20 November 2014
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-andconditions
PREPARATIVE BIOCHEMISTRY, 5(5 & 6), 413-422 (1975)
AN IMPROVED SYSTEM FOR PREPARATIVE STARCH BLOCK ELECTROPHORESIS
P h i l i p C. Kelleher and Carol J. Smith Department o f Medicine, U n i v e r s i t y o f Vermont College o f Medicine Burlington, Vermont, 05401 U.S.A.
Downloaded by [UQ Library] at 02:01 20 November 2014
ABSTRACT
A s t a r c h block e l e c t r o p h o r e s i s system i s described which i n cludes ( a ) a s i n g l e - u n i t L u c i t e e l e c t r o p h o r e s i s chamber, ( b ) temperature c o n t r o l w i t h a c i r c u l a t i n g water bath and c a s t i n g r e s i n coated brass c o o l i n g p l a t e , ( c ) continuous m o n i t o r i n g o f b l o c k surface temperature and ( d ) an easi ly-assembled apparatus f o r r a p i d e l u t i o n o f p r o t e i n from t h e s t a r c h segments. INTRODUCTION Starch block electrophoresis has been used w i d e l y f o r prep a r a t i v e - s c a l e separation o f proteins, b u t t h e apparatus used i s o f t e n make-shift, cumbersome and i n f l e x i b l e , w i t h inadequate cooling.
This r e p o r t describes several m o d i f i c a t i o n s o f t h e o r i g i n a l
method o f Kunkel and S l a t e r 1 which i n c l u d e an enclosed s i n g l e - u n i t electrophoresis chamber, c o n t r o l l e d and constant c o o l i n g o f t h e block w i t h continuous surface temperature monitoring, and a u n i f i e d apparatus f o r r a p i d simultaneous e l u t i o n o f t h e p r o t e i n from the s t a r c h segments.
413 Copyright 0 1976 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 information storage and retrieval system, without permission in writing from the publisher.
KELLEHER AND SMITH
4 14 APPARATUS AND PROCEDURES
F i g . 1 shows a s c a l e drawing o f t h e L u c i t e e l e c t r o p h o r e s i s chamber c o n s t r u c t e d by t h e U n i v e r s i t y o f Vermont I n s t r u m e n t a t i o n and Model F a c i l i t y . 1 of
The two smaller, o u t e r r e s e r v o i r s h o l d 3.5
0.0511 b a r b i t a l b u f f e r , pH 8.6; t h e two l a r g e r , i n n e r r e s e r -
Downloaded by [UQ Library] at 02:01 20 November 2014
v o i r s , 7.0 1 o f 0.2M sodium phosphate b u f f e r , pH 7.5. tubes o f 30mn O.D.
Four U-
g l a s s t u b i n g , w i t h sidearms t o a l l o w f i l l i n g
b y s u c t i o n , serve as s a l t b r i d g e s between t h e b a r b i t a l and phosphate b u f f e r r e s e r v o i r s a t each end o f t h e chamber.
A short
l e n g t h o f rubber t u b i n g i s p l a c e d between t h e phosphate b u f f e r reservoirs t o equalize the b u f f e r levels.
Platinum wire elec-
t r o d e s r u n t h e e n t i r e l e n g t h o f t h e bottoms o f t h e two i n n e r chambers and connect t o two male banana p l u g s molded i n t o one end o f t h e chamber.
The power s u p p l y i s connected t o two
female banana p l u g s which a r e molded i n t o t h e l i d o f t h e e l e c t r o p h o r e s i s chamber, t h u s p r o v i d i n g an i n t e r l o c k system f o r t h e apparatus.
I n t h i s l a b o r a t o r y t h e e l e c t r o p h o r e s i s chamber and
t h e c o o l i n g p l a t e d e s c r i b e d below a r e always k e p t i n a c o n s t a n t temperature room a t 4°C. C o n t r o l l e d c o o l i n g o f t h e b l o c k i s p r o v i d e d by a brass p l a t e which was c o n s t r u c t e d by t h e U n i v e r s i t y o f Vermont Instrumentat i o n and Model F a c i l i t y . 6.4mm O.D.
For t h e c i r c u l a t i o n o f c o o l a n t f l u i d ,
copper t u b i n g was welded t o t h e undersurface o f t h e
p l a t e i n a continuous " S " p a t t e r n .
I n order t o provide a cool-
i n g p l a t e w i t h h i g h thermal c o n d u c t i v i t y , y e t low e l e c t r i c a l con-
415
Downloaded by [UQ Library] at 02:01 20 November 2014
STARCH BLOCK ELECTROPHORESIS
/
FIGURE 1 Scale drawing o f t h e L u c i t e e l e c t r o p h o r e s i s chamber. The g l a s s p l a t e and s t a r c h b l o c k , which r e s t on t o p o f t h e c o o l i n g p l a t e d u r i n g o p e r a t i o n , have been o m i t t e d f r o m t h e drawing f o r t h e sake o f s i m p l i c i t y . Dimensions a r e given i n centimeters.
d u c t i v i t y , t h e brass p l a t e was covered w i t h a c a s t i n g r e s i n ( S t y c a s t No. 2850-KT,
Emerson and Cuming, Inc., Canton, Mass.)
t o f i n a l dimensions o f 63.5cm x 50.2cm x 1.9cm ( w x 1 x t ) . perties o f the r e s i n include:
2 FT /"F/2.54cm;
thermal c o n d u c t i v i t y , 30 BTU/hr/
d i e l e c t r i c constant, 410 V/25.4pm;
t i v i t y , 1015 ohms-cm.
Pro-
volume r e s i s -
The brass p l a t e was p l a c e d i n a mold, and
KELLEHER AND SMITH
416
the liquid resin was poured i n t o form a layer around the plate. The resin was allowed t o harden, and any i r r e g u l a r i t i e s in the surface were corrected by f i l l i n g and hand-lapping.
The plate
res t s on the Lucite buffer reservoir dividers, and the coolant in l e t and o u t l e t tubes extend t h r o u g h grooves cut in one end of the electrophoresis chamber. Water i s circulated through the Downloaded by [UQ Library] at 02:01 20 November 2014
cooling plate by means of a Forma-Temp Model 2220 bath and c i r culator (Forma Sc ie nti fi c , Marietta, Ohio). equi 1i brated a t a temperature of 2°C.
The system i s
After the electrophoretic
separation i s begun, the b a t h temperature i s lowered t o -3°C which maintains the temperature of the upper surface of the block a t 11°C. Block surface temperature i s monitored with a YSI model 42SC telethermometer (Fisher Sc ie nti fi c Co.) with a 4.8mn-diameter surface temperature probe taped t o the polyethylene film covering the block's upper surface. The starch slab i s supported by a 0.64cm-thickY 50.8cmlong and 33.0cm- or 6l.Ocm-wide glass plate framed by Lucite blocks (University of Vermont Instrumentation and Model Facil i t y ) o f various lengths, 5.lcm wide and 2.4cm thick.
The
two Lucite blocks framing the length of the starch slab are 53.3cm long while the other two are 33.0cm o r 61.0cm long, according t o the width of the glass plate.
A sheet of 51um-
thick polyethylene film (Commercial Plastics and Supply Corp., New York, N . Y . )
the same length (50.8cm) as and 30.5cm wider
than the glass plate i s centered on t he moist glass plate.
STARCH BLOCK ELECTROPHORESIS
417
F i l t e r paper wicks are placed along the two widths o f t h e glass p l a t e t o a i d i n t h e d r y i n g o f t h e s t a r c h s l u r r y .
The
t h i c k s t a r c h s l u r r y i s poured i n t o t h i s mold t o a thickness o f 1.25cm and p a r t i a l l y d r i e d w i t h an e l e c t r c fan j u s t u n t i l the s t a r c h slab maintains i t s shape when the L u c i t e frame i s removed.
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A narrow trough i s c u t i n t h e starch w i t h a spatula i n a l i n e perpendicular t o t h e d i r e c t i o n o f m i g r a t i o n and a t a p o i n t between 5.lcm and 17.8cm from t h e cathode end, depending upon t h e expected e l e c t r o p h o r e t i c m i g r a t i o n o f t h e p r o t e i n s .
The
trough i s f i l l e d w i t h the p r o t e i n sample, approximately 0.4 m l / cm o f trough length.
The concentration o f t h e p r o t e i n s o l u t i o n
may be as high as 60 mg/ml, f o r example, whole serum, b u t t h e concentration o f any group o f p r o t e i n s w i t h s i m i l a r e l e c t r o p h o r e t i c m o b i l i t i e s should n o t exceed 30-35 mg/ml f o r optimal resolution.
For separations o f serum proteins, marker samples
o f homologous albumin pre-stained w i t h bromphenol blue may be placed a t t h e o r i g i n , 2.5401 i n from each edge.
A second
sheet o f polyethylene f i l m 48.3cm l o n g i s centered and smoothed over t h e surface o f t h e starch slab.
The s t a r c h slab and glass
p l a t e a r e placed on the c o o l i n g p l a t e and t h e exposed s t a r c h ends covered w i t h b a r b i t a l buffer-soaked c o t t o n t o w e l i n g wicks which extend i n t o t h e b a r b i t a l b u f f e r r e s e r v o i r s .
The wicks a r e
covered w i t h a d d i t i o n a l polyethylene f i l m t o prevent drying, and t h e e n t i r e s t a r c h slab i s wrapped f i r m l y w i t h t h e excess widths o f polyethylene f i l m .
The system i s allowed t o e q u i l i -
KELLEHER AND SMITH
418
brate f o r one-half hour in the electrophoresis chamber with cooling.
The electrophoretic separation i s performed a t a
power supply s et t ing of 600 V, yielding an electr ical f i e l d strength of 8 V/cm, f o r approximately 38 hours or 26 hours for origins a t 5.lcm or 17.8cm respectively from the cathode end. The current i s 90 mA when using the 33.0cm-wide block and 140 mA Downloaded by [UQ Library] at 02:01 20 November 2014
when using the 61.0cm-wide block.
Upon completion of the r u n ,
the starch block i s dried a t room temperature until a small piece maintains i t s shape when held on a spatula.
Forty 1.25cm
sli c es are cut with a spatula, perpendicular t o the direction of electrophoretic migration. Fig. 2 shows the apparatus used t o e lute the proteins from the starch segments.
Each unit consists of a 7.2cm diameter
polypropylene aBuchler funnel (Fisher Sc ie ntif ic Co. ) covered with a 7.0cm Whatman No. 1 f i l t e r paper c i r c l e .
The funnel i s
inserted t h r o u g h a No. 5 1/2 rubber stopper into a 2.7cm I.D. x 17.7cm polypropylene sidearm t e s t tube (Bel-Art Products, Pequannock, N.J.).
The units s i t in a wooden support with
holes of 3.0cm diameter d r i l l e d a t 8.5cm intervals, and a r e interconnected i n se ri e s in an elongated "U" by means of 6.4mm polypropylene T-shaped connecting tubes and 6.4m I.D. vacuum tubing.
The c i r c u i t i s completed by a Y-shaped connecting tube
t o a water vacuum f i l t e r pump. Starch segments a re packed firmly into each funnel with the f l a t end of the plunger from a 50-1111 polyethylene syringe.
A
volume of 0.15M sodium chloride o r suitable buffer a t least 50%
STARCH BLOCK ELECTROPHORESIS
419
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4 20
WLLEHER AND SMITH
greater than the i n i t i a l sample volume i s layered immediately over each packed s t a r c h segment.
When a l l funnels are packed,
vacuum i s a p p l i e d t o t h e tubes and t h e e l u t i o n i s completed w i t h i n seconds. RESULTS AND DISCUSSION Fig. 3 demonstrates t h e e l e c t r o p h o r e t i c separation o f ( a )
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t h e p r o t e i n s present i n a d u l t r a t serum and (b) a preparation o f a d u l t r a t serum macroglobulins.
The packed and e l u t e d s t a r c h
segments were r e - e l u t e d w i t h a second a l i q u o t o f b u f f e r ; no f u r t h e r e l u t i o n o f p r o t e i n demonstrated t h e completeness o f sample removal by t h i s e l u t i o n system.
Recovery o f serum pro-
t e i n s which have been applied t o t h e s t a r c h block i s 100%. Preparative separations o f l a r g e amounts o f complex mixt u r e s o f p r o t e i n s such as these can be achieved more e f f i c i e n t l y by starch block electrophoresis than by column chromatography using an ion-exchange r e s i n such as DEAE-Sephadex.
I n addition,
t h i s s t a r c h block electrophoresis apparatus o f f e r s many o t h e r advantages:
easy assembly and storage, c o n t r o l and m o n i t o r i n g
o f block temperature, f l e x i b i l i t y i n b l o c k s i z e and number o f samples, and r a p i d e l u t i o n of t h e p r o t e i n samples.
The e l e c t r o -
phoresis chamber i s safe f o r t h e operator and f o r personnel worki n g i n the v i c i n i t y since i t i s an enclosed system.
Circulation
o f r e f r i g e r a t e d coolant i s e s s e n t i a l f o r maintaining block temperatures below 15°C; w i t h o u t c i r c u l a t i o n , block surface temperatures may reach 30-35°C.
This p r e p a r a t i v e system i s a l s o
s u i t a b l e f o r use w i t h Geon o r Pevikon as t h e supporting medium.
STARCH BLOCK ELECTROPHORESIS
421
1.50(
1.ooc
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.500 c, h .r
v)
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rc)
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1.
f r a c t i o n number FIGURE 3 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 o f a d u l t r a t serum p r o t e i n s (above) and a d u l t r a t serum m a c r o g l o b u l i n s (below). Arrows on t h e v e r t i c a l axes denote t h e p o s i t i o n s o f t h e electrophoretic origins.
Compared t o s e r i a l single-sample e l u t i o n procedures, t h i s s i m u l taneous e l u t i o n system i s t w i c e as f a s t when performed by t h e same l a b o r a t o r y personnel.
422
KELLEHER AND SMITH
ACKNOWLEDGMENTS The design and t e s t i n g o f t h i s experimental system has been supported by t h e f o l l o w i n g g r a n t s : U.S.P.H.S.
U.S.P.H.S.
Grant GM 11547;
Grant HD 03483; Vermont H e a r t A s s o c i a t i o n Grant AG 63;
American Cancer Society, Vermont D i v i s i o n , Grant #5; U n i v e r s i t y o f Vermont General Research Support Fund; Damon Runyon Memorial
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Fund f o r Cancer Research Grant DRG-1184.
We wish t o thank Mr.
George L u h r and t h e s t a f f o f t h e U n i v e r s i t y o f Vermont I n s t r u mentation and Model F a c i l i t y f o r s e l e c t i o n o f t h e c a s t i n g r e s i n used i n f a b r i c a t i n g t h e c o o l i n g p l a t e and f o r c o n s t r u c t i o n o f t h e apparatus. REFERENCES
1.
H.G.
Kunkei and R.J. S l a t e r , Proc. SOC. E x p t l . B i o l . Med.,
80, 42-44 (1952).
