Silver staining is a difficult technique, that doesn't always give the expected results. I therefore have moved to fluorescent staining (doi:10.1007/978-1-59745-542-8\_14). The method is easy to perform, as sensitive as silver and, if one makes the reagent oneself, very cheap.

The bottom line you mentioned is most likely the front, btw. That is normal.

Hi Engelbert Buxbaum

I prefer to put the gel with developer solution in the microwave for 10 seconds for three times. It well help to introduce and penetrate the stain into the gel. then take more time when you destain the gel in destaining solution.

also try to adjust the voltage on less than 90 voltage (running time).

with my best wishes

Al-Alo K.Z.K

Thanks your reply, Al-Alo K.Z.K. Have you got the straight lines that I point it blue direction?

By the way, Engelbert, I appreciate your suggestion. I'll try as I can.

Quang.. Yes I faced like this problem at past.. But I used the comasee blue stain and I solved my problem by this way

If your protocol worked earlier your formaldehyde might be too old (just try the 2-fold concentration of the formaldehyde or better would be to use new) or the washing steps after the sensitizing are too long.

Here is our protocol if you want to try this:

Fixing: In 50 % ethanol (or methanol), 10% acetic acid for 30min

Washing: In dH2O for 2 x 15min

Sensitizing: In 0.005 % Na2S2O3 for 15min

Washing: In dH2O for a few seconds (otherwise you will wash the sensitizing solution away)

Silver staining: In 0.2 % AgNO3 for 15min

Washing: In dH2O for a few seconds (3x)

Developing: In 2 % K2CO3 (high quality!), 0.074 % formaldehyde (add just before use) which usually takes about 1-5min

Stop: In 50mM EDTA for 15min

Washing: In dH2O for 15min (2x)

The dark vertical lines (blue arrow) are probably still remaining AgNO3 crumbs on your gel. Check if the AgNO3 is completely resuspended in the silver staining solution and use 3 x washing (short!) after the silver staining.

Thank you so much, Steffen. my gel is stained well rightnow but still has the vertical line (blue arrow). I will do the washing step after silver staining a bit longer as you said.