I have stained my sds page gel with Coomasie R-250 and destained it. After that, i perform silver stain to detect my protein of interest. But my band did not turn to brown, in fact it still in blue while gel has been browned. Is there anything I should do?
So after electrophoresis done, i fixate the gels in ammonium acetete solution for 30 min, after that stain in R-250 for 45 min, and followed by destaining until it gives me clear band visualisation. For silver stain, i fixate my CBB stained gel in ammonium hydrogen carbonate solution for 30 min and after that i follow EMBL protocol attached below. Please i need help
Hi Andree,
it seems that you have too much background in the gel, while your silver staining is not effective. Have you tried silver staining without Coomassie in the first place? I would recommend to do an SDS PAGE with not important samples and first optimize your silver staining before you apply it after Coomassie. If that looks nice, a combination is possible.
To address these problems with the brown gel, it is very important to use pure water and pa grade, not too old thiosulfate. This decreases the background dramatically. Also you want to reduce your developing time. If you need any more help, feel free to contact me for my protocol, which is a bit different from yours, but works just fine after coomassie.
Best wishes
Hi Janina,
i have used ultra pure water for every silver staining. I have not try silver staining directly after SDS-Page, but i will do. I will also check thiosulfate expiry date.
I can send my silver stained gel if you would like to.
Yes, can i see your protocol? Thank you very much.
Best wishes
Dear Andree,
as silver staining is more complicated, I highly recommend some practice. I will send you a message with my protocol.
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue