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-   -   Silver staining (http://www.molecularstation.com/forum/protein-forum/26583-silver-staining.html)

Chris B. 08-19-2005 01:18 PM

Silver staining
 
Hello,
I need to stain polylisine - free and conjugated with peptide. Coomasie
Brillant Blue fails with free polylysine and - for my suprise - so does
silver.
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.
Any help?
Regards,
Chris B.



heartthrob 08-28-2005 09:06 AM

Silver staining
 
hello,
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.
Good luck!


Andreas Giessauf 08-29-2005 08:48 AM

Silver staining
 


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

possibility).

Best regards,

Andreas Giessauf


Dr Engelbert Buxbaum 08-30-2005 01:24 PM

Silver staining
 
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
options.


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