Can any please one post me back a protocol for generating total cell lysate from adherent macrophages (RAW 264.7 mouse cell line). I need a minimum of 10ug protein and minimum concentration of 100ug/ml per sample. The cells will be grown on tissue culture treated polystyrene 6 well plates, unless this is a bad idea.
I have a non-SDS lysis buffer formulation that I have been asked to use. Apparently the columns are finicky.
What I really want is the physical treatments/disruptions/shearing/sonications/freeze thaws/etc…
Please also state any “to be avoided” type stuff.
Thanks,
Neal
you can follow my lab publications and can get an idea;
Shinde, S. R. and Maddika, S. PTEN modulates EGFR late
endocytic trafficking and degradation by dephosphorylating Rab7. Nat. Commun.
7:10689 doi: 10.1038/ncomms10689 (2016).
Maddika, S. & Chen, J. Protein kinase DYRK2 is a scaffold that facilitates
assembly of an E3 ligase. Nat. Cell Biol. 11, 409–419 (2009).
Maddika, S. et al. WWP2 is an E3 ubiquitin ligase for PTEN. Nat. Cell Biol. 13,
728–733 (2011).
Thank you for this information, my friend and colleague, Swapnil.
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