I am trying to check a purified recombinant protein on SDS PAGE coomasie Blue and silver staining. Before SDS PAGE, I have checked the total protein concentration using lowry and it shows good amount of concentration. But when I run it on SDS PAGE the coomasie blue stain does not show any bands, while the silver staining one shows only 2 bands. I also check the cell lysate before purification but it shows the same result as the purified lane, only 2 bands and not on the desired MW band. How can I resolve the problem? Thank you in advance for your responses.
Nick Gee thank you for responding, the lowry assay showed that I have 300-400 ug/mL, and loaded 30 uL of sample and 10 uL of loading buffer for the coomasie blue stain, and 6 uL of sample for silver staining
First, confirm the Lowry assay result by measuring the reagent blanks (lysis buffer, purified fraction solution without your protein, mobile phases, etc..)...There should not be any interference that leads to inaccurate protein conc measurement...
Secondly, how did you validate the purification scheme (western, page, ELISA, activity, SPR,...)? I mean, was the recombinant protein expression, lysis, target extraction, and the following purification done successfully? and if yes, how did you confirm?
Silver staining suggests it is a kind of conc dynamic range problem because it is more sensitive than coomassie staining but as you indicated the observed masses (bands) are not at the expected MW range, this could be an expression or purification problem also. Therefore I asked for your confirmation strategy during recombinant protein production...
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