ELIPSIS

ship

between

the

OD of the s tai ned band and

t

he concent­

ra ti on of pro tein is l inear

(.r:ruski

&

Narayan 1 968 , 1 974 ) ;

e)

rapid f i x i ng and s tainill[; foll o1-Jing

FAGS

t o res tri c t

p ro tein di ffusi on from the

bands (Davi s e t al . 1 974)

'

f )

cons tancy of

s taining and des ta:i ning condi t i ons

(

1.va tkin &

Miller

1 970 ) ;

g ) s t o rage of

des tained gels

in acetic ac id and in darkne ss t o

preven t bands fe.ding prior to S•::D nn i. ng (Davis

et al. 1 9'(4 ) ;

h

)

the es tab l i s hment

und er

indivi d ua l ci rcums tances of

t he

parti cular dye

bind i ng re l a t i onsh i ps for each protein

to be

quanti fi ed

(

Kruski &

Narayan

1 97 1 ,

Dol

b

y

1 96 1 ) ;

i )

the es tablishment of a s e t pat te rn of 3Ilalysi.l16 the scans to overc ome

the

problems of overl apping peaks and variable backgrounds

(

Kruski & Narayan

1 974 ) .

The me thod was mod H i ed i n the l igh t of the res u l ts of several exper i men t s whi c h ware conducted to try and i mprove the

technique. The technique as us ed is pt'esented in Chapter 3

wi th t he method and nec essary p�arations de tailed in Appendix

I

Laboratory experiment s ..,;ere conduc ted to i nves tigate the effec ts of acrylami d e conc en tration, me thod of destaini ng and l eng th of the d e s tai ning peri od .

2 : 2 : 2 : 1

_{Concen tration 'i'!l.rec gel concentra ti ons ,}

9,1 1

and

1 3i�

oi acryl ill!li d e w i th propor ti onally increased bis­

acrylamide concentrations were evalua t ed . Eight gels of each c oncen tra tion indicated, by sub j ec tive apprai sal , that

1 3;b

ge ls

gave be t te r pro tein separation than

1 1

; ;, which "1-Tas in t urn b e t t er than t ha t obtained usi ng

9/�

gel B . 'l'he

1 37�

gels were

however, too bri ttle and prone to frac ture for ease of handli ng.

Cons equently

1 1 %

gels w e re us ed throw:h,mt the analysis.

2 : 2 : 2 : 2

i ie thod of Gels 'i'\v o alternative me th ods hav e

been d e s cribed .i n the l i tera t ure :·or the removal of exces s s t ain

f rom gels. First, d i ffus i o n , �1ercby the stain not bound to

pro tein i s washed from the gel matrix by fre qu ent changes of destuining fluid

(2%

Ace ti c

Acid).

Secondly,

electro pho resi s , whereby the unbound s tain migra tes from the gel to the positive

elec trode .

An experimen t was conduc te d to d ete rmine whi ch of these me thods would be s t me et the maj or requi rements of the analys i s ,

tha t i s quan t i ta t ivenes s and speed.

Me thod :

1 2

gel s were loaded , run and s tained i den tically , by the me thods out l ined in

Chapt

er

3.

Two groups of

6

gels viere formed and destained by one of the two me thod s :

a )

The 6 r:els <verc

d

est::1i ned i n a

" home-made" cont

·

nuous circul a t i on d.i.ffus er Hith ai r agi t ation.

'rhe circu l a t i on ra te of fluid

( 2 :

A c e t .i. c Acid

)

was 20ml

/

min \Vi th

diffused s ta i n being removed by an i n-line charcoal f i l ter.

Diffusi on des tai ning was con tinued u11.h l i t was considered that the c lari ty of the inter-band gel matched that of the e l ec trical ly

d.es tai.ned gel s.

b

)

'I'he rer:mining

6

gels <ve re

loaded into de stain

i

ng tubes and plnccd in the baths as for runniP..g the gel s . Destaini ng f luid replaced the running buffer.

A

c urrent of

3-}m

Amp pe r tube N a s run unt i l the s tain front jus t c leared the end of the gel.

Both groups o f gels we re s t o red and s canned as described i n

Chapter 3 . The resul ts are presented i n Table

2 : 1

'l'able

2 : 1

Pro tein

BSA

a

Lac

;3

Lgb test BSA a Lac

,3

Lcb o f 'l'v!O l·l e tho:i s of G e l

Elec trical �i ffusl on

Hean Scan Uni ts

8. 3

1 6. 8

82 . 0

t value

2.02

6 'l

. o

12.0

72 . 0 r.., • _{· �} • .J l f,!ll .�. lC<lllC G

NS

( P

>

0.05)

4 . 4 6 ·�··)H!c

(P

_<

0.005)

7 . 73

i l< lHI

( P

_<

0.001 )

Time of Des tai ning �l uc tricnl -

2.8

hr

Dif fusion -

69

hr

BSA

a I,ac ,3 Lgb

Bov ine S e rum Albumi n

Al pha I,ac ta lbumi n Be ta Lac t oF,l o bu l i n

The e l e c trical d e s taining me thod cons i s te ntly

re sul ted i.n higher pro t e i n c o ncen tra ti ons than the gels

des tai ned b y the d i ffus ion method . �1rthermore the t i me taken for d i fiusion d e s t a i ning m eant that the di f:usion techni que was no t a prac tical al tert;ative to elec t r i ca l des taining.

Having e s tab l i shed that el e c t ro phor e t i c destaining was the mos t sui table for the requi rements of the analysi s , i t was

decided to che ck th e ef fec t of pro longed des taini ng by this method.

Davis ( 1 964)

suggested that prol onGed e l ec t ropho re t ic destaini ng

could resul t i n a red

u

c ti on in the amount o f dye bound to a pro tein.

2 : 2 : 2 : 3

Ti me of

Ne thod

Twen ty fou r gel s were prepared , l oaded , run and s tained under identic

a

l c o nd i ti ons and d e s taineJ el e c t ro pho re t i cally as described earl i e r . A t the point of compl e ti on o f des tni ning

6

ge l s were removed ,

3

at random from e�ch bath. Bl ocks of

6

gels

were removed simi larly af ter

t,

1-.\·

and

2�

hours of ex tra

des taining , \-vi th the curren t beine a l t o red lofi th each block t o maintain

3�-mAmp/ tube

throughout the experiment . Gels lfere s tored and s canned as b e f o re and the resul ts are presented in Table 2 : 2 .

I ,

Tab l 8

2: 2

Ef f e c t of Pro longed El ectrophoretic Des taini rl£1 on

Pro t e i n Conc entration

Ex tra Des taining time

(

hr) Pro t ein IG

j;

B:::>A. a Lac

p

Lgb To t a l

0

su 1 � su

26.6.8

23. 0

1 0 . 5 a

1 0 . 7

3 1 . 3 a

3 1 . 5

1 32 . 8

a 1 28.0

203 . 6

_a

1 97 . 8

SU

S can uni ts - mean of

6

gels

* S ignifi can t at P * * S i gnifi can t at P <

0. 05

<

0 . 0 1

su su

�8.8b

1 7. 5b

10.2

9. 3b

28.7b

28 . 7b F . ra t i o

4 . 1 7

*

3.25

* *

3 . 1 7

**

g . 20

� .

LSD

(5%)

4 . 6

1 . 0

2 . 6

9. 2

1 6 . 0

Wi thin rows

(

pro t e i ns

)

supe rscript " a" di ffers s igni fi can tly

( P

<

0. 0 5 )

t o supers c r i p t "b" .

�

I G Immu noglobulins BSA Bovine Serum A l bumin a Lac Alpha Lactalbumin 8 Lgb Beta Lactoglo bulin

Dis

_c

u

_ssi

on Signi fi c ant losses of

n tain occurred from all

In document Maldito y divino: un acercamiento a la poesía de Raúl Gómez Jattin (página 51-64)