I have prepared 12% Tricine gels to detect 2.2 kDa peptide band. I ran the gel but after silver staining I couldn't see any bands!!
I think the gel itself is fine because I use a prestained marker and the bands are clear and they run fine but I can't see any bands from my samples (knowing that I use a high concentration of protein). I followed the recipe of Schagger for silver staining.
The recipe I use for staining solutions:
Fixation solution
Ethanol 50 mL
Acetic Acid 12 mL
dH2O 38 mL
37 % formaldehyde 50 μL
Sensitizing solution
Sodium thiosulfate (Na2S2O3) 10 mg/100 mL dH2O (=15mg Sodium thiosulfate pentahydrate)
Silver nitrate solution
Silver nitrate (AgNO3) 100 mg/100 mL dH2O
Developing solution
Sodium carbonate (Na2CO3) 3 g
37 % formaldehyde 50 μL
Sensitizing solution 2 mL
dH2O 98 mL
Stop solution (100 mL)
EDTA 25 mM
Do you have any idea, what could be the problem so I can detect any protein bands in the samples ?!
Thank you.
Hello Marian,
what is the origin of your sample (e.g., protein extract or purified protein)? In any case the peptide you want to detect is very small. You can increase the concentration of your gel (e. g., 16 %) so the chance of the peptide running out of the gel is lower. If you have a high concentration of the peptide you can also use Coomassie staining to see whether this is working. For some peptides (especially when these are highly concentrated) silver staining can be problematic. Furthermore, check the protein concentration of your sample (e. g., BCA or Bradford assay) to make sure that the amount of protein you load into the gel pocket is correct.
Good luck with your analysis!
Regards, Christian
Silver staining is particularly sensitive to temperature - if the temperature of your Lab. is below 20 oC you can have serious trouble to stain peptides presenting MW below 4 kDa...One way to fix it is to mildly warm the staining AgNO3 solution and the developer solution to 25-30 oC prior to use.
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