Biomedical Engineering Reference
In-Depth Information
A
0.2% Ponceau S (C.I. 27195) (w/v), 3% TCA (w/v) in ddH
2
O
Solutions/Reagents
B 0.1% (w/v) Fast Green FCF, 1% acetic acid (v/v) in ddH
2
O.
Solutions A and B are reusable for several times.
C 0.05% (w/v) copper(II) phthalocyanine-3,4
,4
,4
-tetrasul-
fonic acid, tetrasodium salt (CPTS) in D
D
destaining solution
: 1% acetic acid (v/v) in ddH
2
O
E 2mM HCl
F .5M NaHCO
3
in ddH
2
O
G
20% ethanol (v/v) in H
2
O
Ponceau S Staining
Immediately after electrotransfer the nitrocellulose (NC) sheet is
Reversible staining
submerged into Soln. A by agitation for about 5 min. Then the
sheet is rinsed with water or PBS until the bands appear and the
background becomes white. The NC may be dried, but the color
bleaches over a longer period. The sensitivity is about 100 ng
/
band.
If the stained NC is blocked with a protein-containing solution,
the staining disappears completely. Detection of proteins by an-
tibodies (immunoblotting) is not influenced by the staining with
Ponceau S.
Fast Green Staining
A nitrocellulose sheet is stained with Soln. B immediately after
electrotransfer or dot blotting. Soln. D is used for destaining. The
detection limit is about 20 ng
/
band.
CPTS Staining
Staining or NC of PVDF sheets with CPTS is also reversible.
The NC membrane is agitated for 0.5 min in Soln. C after elec-
trotransfer. Surplus of dye is removed by Soln. E. The detection
limit for the blue-green bands is down to 10 ng
/
band.
For complete destaining the NC sheet is agitated in Soln. F for
15 - 30 min, followed by rinsing with Soln. G to remove traces of
dye.
PVDF membranes are only destained by Soln. G.
References
Bickar D, Reid PD (1992) Anal Biochem 203:109
2.4.5.2 Staining on Nitrocellulose with Colloidal Gold
A
0.3% Tween 20 (w/v) in PBS
Solutions/Reagents
B
0.1% Tween 20 (w/v), 0.02% Carbowax 20 M (PEG 20000) in
10 mM citrate buffer, pH 3.0
C
colloidal gold, diluted 1:1 with Soln. B (preparation of colloidal
gold see Protocol 4.1.11.2)
After blotting, the nitrocellulose sheet is slightly moved in Soln. A
for 15 min. Before incubation with Soln. B for 5 min,thesheetis
