I need to stain polylisine - free and conjugated with peptide. Coomasie
Brillant Blue fails with free polylysine and - for my suprise - so does
I'm using silver staining method from Amersham Technical Manual (as it say -
based on Heukeshoven and Dernick 1985) but they do not describe which
aminoacids are essential for detection.
Maybe, the using of this method is limitted by F.W. of your
polylisine. It cannot apply to a small peptide. You can change another
polylisine with greater F.W.
Chris B. schrieb:
I used already Ag staining method described by Blum et al.
Electrophoresis 8 (1987) 93-99 for protein staining.
Also, this autor didn't describe which residues are necessary.
But there are just two possibilities how the method works:
(1) Ag+ from the fixing solution is reduced to Ag (colloid)
(2) Ag+ reacts with free -SH groups and forms an Ag-S-R complex
In the first case there must be amino acid residues present
that can be oxidized by Ag+ (cysteines and others).
In the second case there must be amino acid residues present
that can form sulfides with Ag+-salts (cysteines are the only
Chris B. wrote:
What is the molecular weight of your peptides? If it is too small, they
will be eluted from the gel during the staining procedure. Cross-linking
with glutardialdehyde may help in this case.
Also, polylysine will have a considerable positive charge, which would
interfere with Ag+ binding. Staining with an amine reactive fluorescent
probe may be the preferred option, either before or after
electrophoresis. Check the Molecular Probes catalog ([Only registered and activated users can see links. Click Here To Register...]) for
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