hi
this is the first time im detecting phosphoproteins. This is the recipe for my lysis buffer:
250ul of 1M hepes
600ul of 5M NaCl
10ul of 1M MgCl2
20ul of 0.5M EGTA
200ul of 1M B-glycerol phosphate
100ul of 0.1 M sodium vanadate
200ul of 0.5M sodlum fluoride
50ul of glycerol
250ul of 20% triton
8.3ml PBS (can PBS be used?)
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from the above cocktail, take out 858ul and mix it with 1ul of 1M DTT, and final 1X protease inhibitor, final 1X phosphatase inhibitor.
It needs NP40 to extract nuclear proteins (most phosphoproteins go nuclear). See lysis buffer recipe in my paper. https://www.researchgate.net/publication/292615481_Mitochondrial_oligomers_boost_glycolysis_in_cancer_stem_cells_to_facilitate_blebbishield-mediated_transformation_after_apoptosis?ev=prf_high
Article Mitochondrial oligomers boost glycolysis in cancer stem cell...
During the western blotting, -if you’re using an anti-phospho-target antibody-, I would avoid all sources of phosphate groups in the buffers (and BSA for blocking, instead of NFDM), to prevent aspecific competition. No matter about the lysis buffer.
While, if you will move to the detection by immunoprecipitation (and if you’re using an anti-phospho-target antibody), I would change also the lysis buffer composition.
That’s only in case you’re using an anti-phospho-target antibody
Hello Iwan - it would be more helpful if you also included your intended final concentration for EACH reagent next to the volume added and stock solution.
TX-100 and NP40 are equivalent
you may also want to include microsystin LR, as some dephospho rylation may still occur in the buffer you propose to use.
how are you adjusting the final pH of your buffer?
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