I do SDS PAGE at our lab and the results with BSA protein look great.
I want to run it with a complex of Polymer and protein. However, everytime I do it, the complex always travels just like the protein by itself. Therefore, there’s no difference in the bands. We have tried no heating, no beta-mercaptoethanol and no SDS for the loading buffer but they made it worse. I’m using 10% SDS for the loading buffer, could the SDS be the reason of the disruption of my complex?
If the polymer is attached by an irreversible covalent bond, it should remain attached to the protein during SDS-PAGE, and you should see an upward shift of the protein band, assuming the polymer is of a significant size. If you see no shift, then you can conclude either that the attachment of the polymer was unsuccessful or the bond was unstable.
If the complex is noncovalent, it is unlikely to be retained during SDS-PAGE or native PAGE because the gel acts sort of like a gel filtration medium, separating the components on the basis of their sizes. In that case you should not expect to see a shift in the mobility of the protein.
I do the similar experiment now. I attach PEG with antibody with non cleavable linker as well as cleavable linker, for the non cleavable linker i get nice band upward but for cleavable band i failed everytime. I use 5% separating gel and no DTT loading buffer. I dont understand how to solve this problem.
my cleavable linker is pH dependent, cleaves lower than pH 7.4.
Is the cleavable linker sensitive to reducing agent and/or heat, as well as pH?
What is the pH of the SDS-PAGE sample buffer?
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