I would like to elute a protein band for a 10 % SDS-PAGE gel. The amount of protein loaded is from a 5ml induced culture, pelleted and mixed with lamelli buffer. I would like to use the protein for in vitro assays. Is it advisable to stain the gels in commassie? Can anyone kindly help me. Thank you.
Staining with Coomassie Blue in methanol-acetic acid would not be advisable because of fixation of the protein in the gel. If you want to stain the gel and still elute the protein, use Colloidal Coomassie Blue and stain it as lightly as possible (or not at all, if possible). The protein can be electroeluted after destaining. However, the protein is denatured by SDS-PAGE, so if you need it to have its native structure, you will have to find a way to refold it. If you need purified native protein, use chromatography to purify it, not gel electrophoresis. That way, the protein has a chance of retaining its native structure, and you won't have to get it out of a gel. Scale up your culture in order to have a decent amount of material to work with.
Staining with Coomassie Blue in methanol-acetic acid would not be advisable because of fixation of the protein in the gel. If you want to stain the gel and still elute the protein, use Colloidal Coomassie Blue and stain it as lightly as possible (or not at all, if possible). The protein can be electroeluted after destaining. However, the protein is denatured by SDS-PAGE, so if you need it to have its native structure, you will have to find a way to refold it. If you need purified native protein, use chromatography to purify it, not gel electrophoresis. That way, the protein has a chance of retaining its native structure, and you won't have to get it out of a gel. Scale up your culture in order to have a decent amount of material to work with.
Laemmli buffer together with SDS-PAGE will denature your protein (lose its 3D structure and activity) so you will have to try different refolding/dialysis protocols once purifying so that it regains activity. As Adam said, chromatography will be a better option. Beware that if it is a mammalian protein it may not fold properly in a bacterial expression system so the protein may be inactive anyway. If you don't have access to chromatography equipment try non-denaturing PAGE. Load two samples - a small amount to stain to determine where your protein is and then second larger sample of your protein that you want to purify.
Proteins separated on polyacrylamide gels can be transferred onto immobilized membranes (blotting). However, in many instances, a gentle method to elute the proteins directly into a liquid phase is required to avoid chemical modification or denaturation of the eluted proteins. Common approaches include elution by diffusion and electroelution.
For getting more details and to have a clear idea you can refer to the following link:-
http://www.pall.in/main/laboratory/literature-library-details.page?id=2273
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