Biomedical Engineering Reference
In-Depth Information
2.4.2.1 Citrate/Formaldehyde Development
A
10% acetic acid (v/v), 30% methanol (v/v) in ddH
2
O
Solutions/Reagents
B 15% methanol (v/v) in ddH
2
O
C 5% glutaraldehyde (v/v) in ddH
2
O (usable for several times)
D .7ml of 25% ammonia and 2.0 ml 1 N NaOH are filled up to
16.0 ml with ddH
2
O, then 2.0 ml 10% AgNO
3
(w/v) are added
dropwise with shaking. The precipitate formed by a drop has
to dissolve completely before giving the next. If the last drops
are not dissolved completely, some ammonia can be carefully
added. The resulting solution is pale brown. When the addition
of silver is complete, ddH
2
O is added to 100 ml.
The solution is prepared freshly before using. After use, the
solution has to be destroyed by hydrochloric acid.
E
reduction solution
: 0.025 g citric acid and 0.185 ml 27% form-
aldehyde in 100 ml ddH
2
O (stabilize for a longer period if
frozen)
F
0.5% acetic acid (v/v) in H
2
O
The gel to be stained should not be thicker than 1 mM for that
diffusion of the reactants is quickly. The volumes of used solutions
should be at least the tenfold of the gel volume.
The gel is moved gently during all steps. Especially immediately
before, during, and after the application of Soln. D the gel must not
be touched with unprotected fingers (use gloves and/or tweezers)
or come in contact with chloride-containing solutions.
After electrophoresis the gel is fixed in Soln. A at least 20 min.
Then it is incubated three times in Soln. B for 20 min each, 30 min
in Soln. C, and three times in Soln. B again. Incubation in Soln. D
succeeds for 30 min. Excess of glutaraldehyde is removed by three
washings with ddH
2
Ofor5min each.
Development is done with Soln. F. Pouring off Soln. F and add
Soln. G when first bands appear to stop the reaction.
Overstained gels must be freed from acidic and/or silver
residues by washing with several changes of ddH
2
O. Then the stain-
ing can be reduced or removed according to Protocol 2.4.2.7. A new
silver stain is possible, but you never get the same picture as at the
first time; therefore, the weakening should be done only if abso-
lutely necessary.
A second staining with Coomassie Brilliant Blue following the
silver stain perhaps produces bands of proteins not or weak stain-
able by silver, because silver staining is, on the one hand, more
sensitive than other procedures, but on the other hand, also not
universal.
For a second staining with Coomassie, the gel is equilibrated
Second staining
three times in Soln. F and then stained as described in Proto-
col 2.3.1.2.
Sometimes the electrophoresis buffer produces a high back-
ground. A significant, rapid increase of electric field strength, as
