Hi everybody!
I'm workin with outer membrane vesicles from E. coli (OMVs) and I want make SDS-PAGE, however I can´t see nothing in Coomassie staining. I use this protocol:
1. OMVs are isolated from E. coli by ultracentrifugation and resuspended in PBS.
2. Protein concentration is quantified by Bradford assay and I use 50ug of protein for SDS-PAGE.
3. The samples are mixed with B-mercaptoethanol, buffer load 5X and H2Oddi.
4. This samples are heated to 90°C for 5 minutes and finally, the samples are load in the polyacrilamide gel (12%). I use 80V/30 minutes and 150V/60 minutes.
5.Finally I fix with acetic acid for 1 hora and coomassie for 20 minutes...
Regards!!!
Protein concentration is quantified by other method more sensitive from Bradford assay and used the Coomassie Blue R-250 for stain ...
Are you using the right Coomassie stain for gels (R, not G)? The gel should be stained with Coomassie Blue R-250 dissolved in a mixture of methanol and acetic acid. Fixing before staining is not necessary. You also must destain after staining to clear the protein-free parts of the gel. Try putting in a standard protein, such as BSA, as a positive control to make sure the staining procedure is working.
Hi
Absolutely I agree with dear Shapiro, but there is more thing that I think about other issue.
Is your concentration enough for stainig with coomassie or you have to stain with silver
Protein concentration is quantified by other method more sensitive from Bradford assay and used the Coomassie Blue R-250 for stain ...
Thank's everyone!... I prepared new Coomassie stain and works perfect!...
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