Biomedical Engineering Reference
In-Depth Information
Stainthegelfor1-3h. Further comments see Protocol 2.3.1.1.
Staining with Fast Green is partial suitable for quantitative
densitometry at 625 nm.
2.4.1.5 Stains All
(4, 4
,5,5
-Dibenzo-3,3
- diethyl - 9 - methylthia carbocyanine bro-
mide)
stock solution
:0.01g dye is dissolved in 10 ml pure formamide.
Solutions/Reagents
A
It is stored airtight in the dark.
A
working solution
: Immediately before using, Soln. A is diluted
1:20 with Soln. B. The working dilution can be used only once.
B
50% formamide (v/v) in deionized water.
Acidic fixed gels are equilibrated three times for 20 min in Soln. B.
Then the gel is stained with Soln. A
for 1 h in the dark followed by
destaining in Soln. B.
Stains All is light-sensitive and bleaches slowly at the air. The
stained gel should dry in a vacuum gel dryer immediately after
destaining and documentation.
The main advantage of Stains All are different colors produced
by proteins and nucleic acids.
2.4.1.6 Staining of Proteolipids, Lipids, and Lipoproteins
A .5g Sudan Black B (Solvent Black 3, Fat Black HB, C.I. 26150)
Solutions/Reagents
is dissolved in 20 ml acetone, and then 15 ml of glacial acetic
acid are added followed by 80 ml ddH
2
O. The solution is cen-
trifuged for 15 min at RT with 3000
×
g, the supernatant is
used for staining.
B
15% acetic acid (v/v), 20% acetone (v/v) in ddH
2
O.
After electrophoresis the gel is fixed either in the common acetic
acid/methanol/H
2
O mixture or in Soln. B, but never in aqueous
TCA.
Staining is done by incubation with a tenfold gel volume of
Soln. A overnight. Use several changes of Soln. B for destaining.
Remaining colloidal dye on the surface of the gel is wiped off with
a wetted tissue.
References
Maurer HR (1971) Disc electrophoresis, p. 76, W. de Gruyter, Berlin
2.4.2 Silver Staining of Proteins in Gels
The following procedures are well improved and easy to perform.
Nevertheless, in some cases variations of the protocols concerning
silver binding and/or reduction can give better or other results, be-
cause proteins bind silver differently and are observed with varying
colors. Some hints are given in the cited papers.
