Hello!
I am working on a protocol to extract as many native proteins as possible from brain tissue.
The end goal is to use the supernatant of the lysate in immunoprecipitation against some antibodies with unknown targets.
Since the epitope of the unknown targets could be non-linear, I want to keep as many proteins as possible in their native state, while still extracting as many proteins as possible from the tissue sample.
Has anyone tried to make such a lysis buffer before?
I am leaning towards dissolving the sample in two fractions: one with a milder detergent (0.5 - 2% DDM, for a higher chance of conformational stability of membrane proteins) and a second fraction with a slightly harsher detergent to dissolve proteins that are harder to solubilize (I am thinking CHAPS, maybe with a low concentration of SDS?)
Also, how much detergent do you usually include in IP wash/elution buffers to prevent precipitation? Would 2-3 times higher than the CMC be enough here?
Thank you!
Johanna
https://www.hopaxfc.com/en/blog/6-biological-buffers-recommended-for-protein-purification
https://hrcak.srce.hr/file/120419
https://bmcresnotes.biomedcentral.com/articles/10.1186/s13104-020-4929-1
https://www.sciencedirect.com/science/article/pii/S1319562X16300237
https://journal.ipb.ac.id/index.php/tasj/article/download/27490/20782/
https://onlinelibrary.wiley.com/doi/10.1002/pmic.201300239
Regards
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