Special greeting. I have a protein extraction solution containing 1% SDS, 300 mM B-mercaptoethanol, 100 mM Tris and 8 M urea, 10 mM EGTA. Is there a method that allows me to measure protein concentration without precipitating or filtering, considering that this solution has interferents for all colorimetric techniques (Bradford, Lowry, BCA)?
You could run the sample on SDS-PAGE, stain the gel with Coomassie Blue, and measure the amount of stain in the lane using a densitometer or gel imager. You could then compare the amount of stain to the amount in some protein standards run on the same gel.
Its difficult to test concentration of protein with interferences but you can try Biuret assay by diluting your sample.
Hi,
Like Adam suggested above, similar to that I could suggest a more like qualitative approach I use when I am want to load sample for initial gel run and I want to load sample almost equally. You can take a whatman paper, make two lanes, and make several circles usinga pencil. In one lane add at least 5ul of your unknown quanity of protein sample and in another lane load several conc. of known BSA samples. Let the samples dry and then you can do the coomassie blue staining followed by destaining. Once everything is destained except the circles containing protein, you can either visually or using image you can find out intesnity and by that you can estimate the concentration of unknown protein using known BSA conc. you loaded in second lane.
What a good idea, thank you all for your answers.
Best regards.
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