Hi,

I'm a research student and I just recently started to silver stain my gels using a kit from BioRad but I came across some problems:

1. I tend to get a dark background eventhough I stopped the development on time as stated on the kit.

2. I get a darker background on the top of the gel than at the bottom. the sample is whole virus at 30 micrograms per well.

3. I used commassie pre-stained ladder, and only 3 out of 8 bands are visible. sometimes it's just a big mess with a dark smear from top to bottom. i thought it was just a matter of loading too much ladder, but when i diluted it with sample buffer, the result was still the same.

4. sometimes the bands are only visible after a long time and by then the background was already too dark.

I use precast 4-20% gels, added 5% BME to sample buffer.

Can anyone help me?

Thanks a lot!

1. I tend to get a dark background eventhough I stopped the development on time as stated on the kit.
free silver not totally removed, instead of do one 15-30 sec wash after the silver treatment, do three times wash with a total of 15-30 sec.
and do good wash for other steps to reduce background

2. I get a darker background on the top of the gel than at the bottom.
did you recycle your gel running buffer? use new buffer every time. if not recycling, check your running buffer

3. I used commassie pre-stained ladder, and only 3 out of 8 bands are visible. sometimes it's just a big mess with a dark smear from top to bottom. i thought it was just a matter of loading too much ladder, but when i diluted it with sample buffer, the result was still the same.
seems you have overloaded. it still happened after the dilution, you may have touched the bottom of the well when loading

and do a longer fixation


these should solve your problem

Hi,

Thanks for the tips. I'll try also to wash the glassware with nitric acid. hopefully I'll get better result next time.

Anggia