3. CONTEXTO CONCEPTUAL Y NORMATIVO EN EL CUAL SE DESENVUELVE
4.2 CONCEPCIÓN QUE TIENE EL REVISOR FISCAL, SOBRE LA
GEL PREWGTH
%
- VOL OF 30^ ACRYL SOLUTICW (I)(m l) VOL OF VOL OF 10^ SEPARATION SDS (ml) BUFFER- (g) (ml) VOL OF WATER (m l) VOL OF TEMED f k l) VOL OF APS (m l) 6 10 0 .5 6 .2 5 33 <000 10 0 .2 5 9 15 0 .5 6 .2 5 28 LLtQ 10 0 .2 5 12 20 0 .5 6 .2 5 23 10 .0 .2 5 15 25 0 ,5 6 .2 5 18 10 0 .2 5 18 30 0 .5 6 .2 5 13 10 0 .2 5 21 35 0 .5 6 .2 5 8 10 0 .2 5 24 40 0 .5 6 .2 5 3 10 0 .2 5The
9fo
s e p a ra tio n g e l we m ostly used was made my m ixing15ml o f 30^ acry lam id e s o lu tio n ( i )
0.5ml o f 10^ (w/v) SDS
6 .2 5 s e p a ra tio n b u ffe r (g) and 28.0m l w a te r..
The m ix tu re was degassed f o r 2 m inutes b e fo re adding 1 ( ^ o f TEMED and 0.25ml o f 10^ (w /v) ammonium p e rs u lp h a te . The m ix tu re was poured in to th e mould to a le v e l 20mm below th e bottom o f th e n o rch . The m ixture was o v erlay ed w ith about 1ml of w a te r. The m ixture was l e f t to polym erise f o r 2 h o u rs, a f t e r which th e w ater was removed and th e n
stacIdLng g e l added t o f i l l th e g e l mould. F ive p er c e n t s ta c k in g g e l was p rep ared by m ixing
lOml o f IC^ acrylam ide s o lu tio n ( j ) 0.2m l of 10^ (w/v) SDS
4.8m l o f stacIdLng b u ffe r (h) and 4.8m l o f m t e r .
The m ix tu re was degassed and th e n 10/^1 o f TEMED and 0.2m l of 10^ (w/v) ammonium p e rsu lp h a te were added t o achieve p o ly m e risa tio n . The m ix tu re was o v erlay ed w ith a p a s te u r p ip e tte f u l o f w ater and l e f t to polym erise f o r 1 h o u r. The f i r s t dim ension g e l ( is o e le c tr i c fo c u sin g g e l) was
th e n loaded in to th e SDS g e l and s e a le d w ith 1 $ (w/v) a g a ro se , mixed w ith 0.01$!^(w/v) bromophenol b lu e made in sam ple b u ffe r (k ). The low er tan k o f th e e le c tro p h o re s is a p p a ra tu s was f i l l e d ifith e le c tro d e b u ffe r (L ), th e p la te s loaded and th en th e to p ta n k was f i l l e d w ith th e same b u ffe r. E le c tro p h o re s is was ru n a t 4mA per p la te u n t i l th e tra c k in g dye (bromophenol b lu e ) was ab o u t fmm from th e bottom of th e g e l.
5.4 .6 D etectio n of p e p tid e s
A fte r th e run., th e g e l was removed from th e p la te s and fix e d w ith
15^ (w/v) tr ic h lo r o a c e tic a c id (s o lu tio n O) f o r
JO
m in u tes. The g e l was th e n s ta in e d f o r 2 hours w ith Goomassie b r i l l i a n t b lu e R250s o lu tio n (P ). The g e l was f i n a l l y d e sta in e d by soaking in s e v e ra l changes o f d e s ta in in g s o lu tio n (g) and th e p ro te in p a tte rn o b ta in ed
41
blood
sp in a t 500% f o r 10 m inutes > V
RED blood cells
Wash 3X in O .I5M PBS pH8
and s p in a t jOOOg f o r 10 m inutes each tim e .
i
RED blood cells
Lyse 2X in 5 ^ sodium phosphate b u ffe r PH8 and s p in a t 10 OOOg f o r 10 m inutes each tim e,
Ÿ
RED blood cell GHOSTS
Wash in lOmM T ris-H G l b u ffe r pH8 and sp in a t 10 OOOg f o r 10 m in u tes.
RED blood cell GHOSTS
Suspend in 56mM sodium b o ra te b u ffe r PH8 and sp in a t 10 OOOg f o r
10 m in u tes.
Ÿ
RED CELL GHOSTS
Suspend in 56mM sodium b o ra te b u ffe r pH8 and sp in a t 3 0 OOOg fo r
10 m in u tes.
RED CELL MEMBRANES (F in a l P e lle t) FIGURE 3
Flow diagram f o r p re p a ra tio n of re d c e l l membranes.
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