i am using sds page to know the molecular weight of protein i am working with. i have used coomassie blue to stain the gel.
Its been over a week now that i am changing the destaining solution every day but i see no change in the gel.
There are molecular markers visible.. and protein is in form of a smear and not bands...further the gel is yet blue so how do i speed up this process?
I don't know the specifics of what you've done, but some general advice to speed up destaining of SDS-PAGE:
Gently agitate the gel. Rocking will speed up destaining.
Add a KimWipe (or similar lint-free lab wipe) to the destain. Make sure the Kimwipe is scrunched up in a corner and NOT on top of the gel (it can stick to the gel). The wipe will "suck up" a lot of Coomassie blue. You can remove the old and add a new wipe. This is helpful for those of us who've forgotten to destain a gel and have left it overnight in stain.
I know some people who gently heat the destain, but please use care, particularly if you have methanol or ethanol in the destain. You might microwave for A FEW SECONDS. Be safe if you try this and don't set anything on fire or breathe nasty fumes. Personally, I don't think this is a good idea, but heat will speed up destaining.
If a gel isn't destained after several changes, you might be staining it too much. In the future you might try using less stain (mix the stain with some destain). I've been recently using BioRad BioSafe stain, and someone just showed me a trick - rather than using full-strength stain, we use 1/2 or 1/4-strength stain overnight. Stains nicely, and destaining is more efficient.
Smearing is a whole different problem, and makes me wonder if your stain isn't fixing your gel. The classic acid-alcohol-Coomassie blue stain both stains and fixes the proteins in the gel. If you're not using the right stuff, your bands will diffuse. What, exactly, are you using to stain and destain your gel? And, you should take the gel down immediately after turning the power off, and fix/destain promptly. Leaving a gel sitting without current running will let diffusion occur.
Post a picture of your next gel, please.
Dear Mr. Sharma
Please check your destating solution, you can check for the compsition of destating solution in protein protocol hand book by Walker. As far as your smearing issue is concerned, you need to take care of few points verry seriously. If you are Isolation you protein of interest use protease inhibitors for storing your protein samples. Check the pH of both (0.5M and 1.5M)Tris-HCl buffer you are using to prepare your gel and store them property. Use freshly prepared running buffer to run electrophoresis.
Goodluck
Hello Himanshu,
We are very happy users of the protocol described in this publication. In-fact after trying this protocol, we never stained our gels using the traditional method. If you have Coomassie Brilliant Blue G-250 available in your lab, I'll suggest you to give this protocol a try.
Staining of proteins in gels with Coomassie G-250 without organic solvent and acetic acid. http://www.ncbi.nlm.nih.gov/pubmed/19684570
This protocol will not help with your smearing problem (could be several reasons for that) but sure will help with speedy staining and destaining of the gels.
I don't know the specifics of what you've done, but some general advice to speed up destaining of SDS-PAGE:
Gently agitate the gel. Rocking will speed up destaining.
Add a KimWipe (or similar lint-free lab wipe) to the destain. Make sure the Kimwipe is scrunched up in a corner and NOT on top of the gel (it can stick to the gel). The wipe will "suck up" a lot of Coomassie blue. You can remove the old and add a new wipe. This is helpful for those of us who've forgotten to destain a gel and have left it overnight in stain.
I know some people who gently heat the destain, but please use care, particularly if you have methanol or ethanol in the destain. You might microwave for A FEW SECONDS. Be safe if you try this and don't set anything on fire or breathe nasty fumes. Personally, I don't think this is a good idea, but heat will speed up destaining.
If a gel isn't destained after several changes, you might be staining it too much. In the future you might try using less stain (mix the stain with some destain). I've been recently using BioRad BioSafe stain, and someone just showed me a trick - rather than using full-strength stain, we use 1/2 or 1/4-strength stain overnight. Stains nicely, and destaining is more efficient.
Smearing is a whole different problem, and makes me wonder if your stain isn't fixing your gel. The classic acid-alcohol-Coomassie blue stain both stains and fixes the proteins in the gel. If you're not using the right stuff, your bands will diffuse. What, exactly, are you using to stain and destain your gel? And, you should take the gel down immediately after turning the power off, and fix/destain promptly. Leaving a gel sitting without current running will let diffusion occur.
Post a picture of your next gel, please.
U will see magic when u use water and acetonitrile destaining solution. I ideally use 30 % acetonitrile in water. I made it when our last methanol stock was out. How ever not sure if it has any impact on the downstream experiments like ingel digestion and maldi
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