Biomedical Engineering Reference
In-Depth Information
µ
µ
B
600
l 0.1% H
2
O
2
-urea adduct (w/v) in ddH
2
O, 10
l 10%
µ
CuSO
4
(w/v), 5
l 10% NiSO
4
(w/v) or NiCl
2
and 9.35 ml PBS
are mixed, then 40
µ
l of Soln. A are added. Prepare reagent
immediately before use.
C .4mg
/
ml (20 mM)3,3
,5,5
-tetramethylbenzidine (TMB) in
DMSO (stock solution).
D .2M sodium acetate pH 5.0.
E 0mM ammonium molybdate ((NH
4
)
6
Mo
7
O
24
·
4H
2
O, MW
1235.86) in ddH
2
O. Stable at RT.
F .1%H
2
O
2
-urea adduct (10.6 mM H
2
O
2
) in ddH
2
O. Stable for
several days in a refrigerator.
G
precipitating TMB
: mix Soln. C, D, E, F, and ddH
2
Oinaratio
of 0.5:5:1:2:1.5 prior to use
24
H .1M H
2
SO
4
4-Chloro-1-naphthol (4-CN) and diaminobenzidine (DAB) are
used in Soln. B; staining with tetramethylbenzidine (TMB) is done
in Soln. G at RT for 10 - 20 min.
After color development Soln. B and G, respectively, are poured
off and the membrane is rinsed intensively with water.
When precipitating TMB is used, a visual intensification is pos-
sible by shaking the membrane in Soln. H for some seconds. The
blue color of bands switches to brown and the background becomes
paler.
Stained blots are dried on air on a sheet of filter paper. If ra-
dioactive compounds are also transferred, autoradiography of the
dry membrane is possible at −70
◦
C.
2.5.4.2 Detection Using Alkaline Phosphatase (AP)
A
100 mM Tris, 100 mM NaCl, 5 mM MgCl
2
,pH9.5
Solutions/Reagents
µ
B
37.5
l of 5% 5-bromo-4-chloro-3-indolyl phosphate (X-phos-
phate, BCIP) (w/v) in DMF and 50
µ
l of 7.7% nitroblue tetra-
zolium chloride, toulidinium salt (NBT) (w/v) in 70% DMF
(v/v), added and mixed immediately before use
Alkaline phosphatase cleaves phosphate esters at medium al-
kaline pH in the presence of magnesium ions.
When AP will be taken for detection in Western blots, after
antibody processing the membrane is equilibrated twice for 5 min
each in Soln. A. Equilibration buffer is poured off and the color is
developed in Soln. B completely covering the membrane. During
this step the membrane should be moved very gently only.
Visualization Soln. B is poured off after 5 - 15 min, and then the
membrane is washed for some seconds with water and dried on
air covered by filter paper. Since the dye is air- and light sensitive
and bleaches over a longer period, the dry membrane should be
wrapped with a foil and stored protected from light.
24
Precipitating TMB ready to use is offered by several companies.
