I am performing reaction between iodoacetyl-PEG-biotin (linker) and papain. Thiol group of papain reacts with the linker to form a thioester bond. then I use streptavidin beads to fish out papain bound to linker. So in order to use minimum amount of streptavidin beads I need to know minimum amount of papain I could see in a SDS PAGE gel. Can anybody please help?
(1 mg of streptavidin beads can bind 400 pmol of biotinylated peptide.)
Using silver stain, you can detect less than 1 ng of protein. If using coomassie blue G-250 or R-250, you can visualize 10 and 100 ng of protein respectively.
The detection limit depends on the quantification method. Proteins can be visualized by Coomaassie G250, Coomassie R250, Silver Stain, trihalo methods (e.g. Biorad's stain free), and probably others. Each has a different sensitivity.The sensitivity also depends on the type of gel used, and whether the gel can reasonably resolve the target protein (I've found gradient gels give better resolution usually), and the protein purity.
In general you can detect a single protein with R250 with as little as a few hundred nanograms if it's pure. However, the assay isn't linear at the detection limit. You need to load closer to 1ug before you're in the linear range, and too much protein again exceed the linear range.
As such, you'd be well advised to test this empirically. Papain is cheap (depending on grade), as are SDS-PAGE gels and the staining methods.
depends how you develop the gel and what you want to do with the protein afterwards ...silver stain is very sensitive if done well for visualizing
silver stain donw to less than a ng but cant use the proteins for anything else like MS afterwards
Coomassie down to maybe 100 ng but can do other things.
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