34
L. CASTELLANOS-SERRA et al.
completely transparent. Renew the chelating reduction buffer and continue incubation for an additional 30 mins. To S-pyridylethylate, add 4-vinylpyridine (2 %
vlv) in the reduction buffer containing the gel and incubate in the dark, at 25°C
for 60 mins. After incubation, halt the alkylation by adding ~-Mercaptoethanol
(2 % vlv) directly to the solution, then discard the solution and wash the gel band
extensively in water (3 x 1ml) for 10 mins
Comments
i) Protein fIxation through zinc-mediated aggregates should be fully reverted
before (or during) any procedure that requires proteins to be removed from
the gel or accessed by enzymes or chemical reagents. This step is called 'protein mobilization'; during this step, protein losses from the gel band due to
diffusion with the chelating solution (a,b,d) are only 10 % or less (determined with radioactive MW markers) (Castellanos-Serra, unpublished
results) while losses due to diffusion in citric acid are signifIcantly lower.
ii) The chelating agent should be chosen to best suit the next analytical step: (a)
or (b) for fast passive elution or electrotransfer; (a) for microdeglycosylation and in-gel renaturation; (b) for in-gel digestion and electroelution; (d) for protein reduction and alkylation during zinc chelation.
iii) Incubation in citric acid usually gives very clean fIrst cycles upon automatic
sequencing, nevertheless, some proteins tend to give lower transfer yields to
membranes in comparison with those treated at neutral to basic pH.
iv) Incubation in EDTA or glycine, in general, gives very high yield on transfer
and elution. Nevertheless, in one case, we found disappointingly low yields
on transfer when a protein was mobilized in EDTA. This particular protein
was a Ca (H)-binding venom toxin, that probably self-aggregated on the gel
after removal of the metal.
v) Thiol reagents (2ME, DTT, DTE) are very effective zinc chelators; proteins
can be reduced during protein mobilization by using conditions described
under (d). For an effIcient reduction, increase, if necessary, the incubation
time and the temperature (i.e., 56°C, 2 x 10 mins), next follow the procedures
for in-gel alkylation with 4-vinyl pyridine or other alkylating agents (Moritz
et aI, 1996b).
vi) After mobilization, proteins can be then electroeluted or digested following
conventional procedures. Nevertheless, standard electro elution devices with
dialysis membranes may lead to variable, generally low recoveries when
working at the low picomole level, as losses associated to adsorption on
membranes can be signifIcantly high. In cases such as these, the fast passive
elution procedure (see 2.6) is recommended, yielding almost quantitative
recoveries at the low picomole range.
L. CASTELLANOS-SERRA et al.
completely transparent. Renew the chelating reduction buffer and continue incubation for an additional 30 mins. To S-pyridylethylate, add 4-vinylpyridine (2 %
vlv) in the reduction buffer containing the gel and incubate in the dark, at 25°C
for 60 mins. After incubation, halt the alkylation by adding ~-Mercaptoethanol
(2 % vlv) directly to the solution, then discard the solution and wash the gel band
extensively in water (3 x 1ml) for 10 mins
Comments
i) Protein fIxation through zinc-mediated aggregates should be fully reverted
before (or during) any procedure that requires proteins to be removed from
the gel or accessed by enzymes or chemical reagents. This step is called 'protein mobilization'; during this step, protein losses from the gel band due to
diffusion with the chelating solution (a,b,d) are only 10 % or less (determined with radioactive MW markers) (Castellanos-Serra, unpublished
results) while losses due to diffusion in citric acid are signifIcantly lower.
ii) The chelating agent should be chosen to best suit the next analytical step: (a)
or (b) for fast passive elution or electrotransfer; (a) for microdeglycosylation and in-gel renaturation; (b) for in-gel digestion and electroelution; (d) for protein reduction and alkylation during zinc chelation.
iii) Incubation in citric acid usually gives very clean fIrst cycles upon automatic
sequencing, nevertheless, some proteins tend to give lower transfer yields to
membranes in comparison with those treated at neutral to basic pH.
iv) Incubation in EDTA or glycine, in general, gives very high yield on transfer
and elution. Nevertheless, in one case, we found disappointingly low yields
on transfer when a protein was mobilized in EDTA. This particular protein
was a Ca (H)-binding venom toxin, that probably self-aggregated on the gel
after removal of the metal.
v) Thiol reagents (2ME, DTT, DTE) are very effective zinc chelators; proteins
can be reduced during protein mobilization by using conditions described
under (d). For an effIcient reduction, increase, if necessary, the incubation
time and the temperature (i.e., 56°C, 2 x 10 mins), next follow the procedures
for in-gel alkylation with 4-vinyl pyridine or other alkylating agents (Moritz
et aI, 1996b).
vi) After mobilization, proteins can be then electroeluted or digested following
conventional procedures. Nevertheless, standard electro elution devices with
dialysis membranes may lead to variable, generally low recoveries when
working at the low picomole level, as losses associated to adsorption on
membranes can be signifIcantly high. In cases such as these, the fast passive
elution procedure (see 2.6) is recommended, yielding almost quantitative
recoveries at the low picomole range.