Hello all,
The protein I'm purifying seems to destain very quickly on Coomassie stained SDS-PAGE gels (red box in image). I've heard that proline-rich proteins (like this protein) have a propensity to stain differently but wasn't aware that they would so readily disappear.
I was wondering if anyone else has experience of this? and secondly can anyone suggest a different stain that should work better - I dont like that idea of routinely silver staining/blotting my gels!
Thanks in advance.
Take a look at any of the negative staining protocols available online. They are fast, reversible, more sensitive than Coomassie and don't suffer from protein-to-protein variations (although contrast is never that good). I am partial to this one:
Article "Reverse-staining" of biomolecules in electrophoresis gels: ...
Another alternative is fluorescent staining, if you have the dye and the instrumentation. Sensitivity is high and dynamic range is huge.
Regarding Coomassie staining of proline-rich peptides yes, I do remember from some time ago the H8 antigen from Neisseria sp. It did not stain well using Coomassie, if at all.
Hope it helps!
Does it fully destain or reach an equilibrium. I am wondering if Coomassie is partially binding to your protein non-specifically, a situation that I have not encountered myself.
Binding of Coomassie blue to basic amino acids is much more efficient then to acidic amino acids. Glycosylation usually provides negative charge to protein, that is why highly glycosylated protein is difficult to be stained by Coomassie Blue.
You can also perform glycoprotein staining and compare with your CBB stained gel and look at the glycosylated proteins.
This links might help..
https://www.users.miamioh.edu/hagermae/Coomassie%20Stain%20for%20PRPs%20on%20SDS%20Gels.pdf
https://info.gbiosciences.com/blog/how-to-stain-glycoproteins-in-polyacrylamide-page-gels
Thanks for the replies all! Arthur Lustig is does destain completely if left for long enough (probably led to me throwing away quite a few protein preps).
I read that coomassie binds to basic residues and this protein has got aquite a few Arg residues so I'm unsure why it does destain so readily. Its got quite a high pi (around12) and I was wondering if this would contribute to its destaining properties.
It might be that your proteins are not fixed in the gel and migrate out of it. John Palmer how does your staining procedure look like?
Kristian Arambasic stain for 40 mins in 50% Methanol 50% (v/v) Glacial acetic 10% (v/v) and G250 0.125% (w/v). Why do you think this one protein wouldn't fix but the others would?
- Badges
- Science topic
- Similar topics
- Gels