Whole cell lysis of fibroblasts - (May/21/2010 )
i had two questions regarding whole cell lysis of my fibroblast pellet I would really welcome your advice.
1) When re-suspending the pellet in lysis buffer, do you have to incubate at RT for a while for lysis to take place, or is resuspension enough.
Usually what I do after resuspending is sonication in an ice bath and then centrifug. to collect the protein solution (supernatant).
2) My lysis buffer recipie is 0.02M Tris, 0.1 M NaCl, 1mM EDTA and 10% Triton X 100 (pH 7.4).
I have tried to use the Micro BCA Assay for protein quantification and find that my blank turned purple (blank: only lysis buffer + working reagent).
This should not happen, as there should not be anything reducing the Cu2+ in the working reagent to Cu+ (the latter interacts w/ BCA to produce color).
I was wondering what you thought is causing this purple color? Could the 10% Triton X 100 be the problem? the Instruction manual says the assay is compatible w/ 5% Triton X
but I can't see how the Triton may be reducing the Cu2+.
Yours ideas are most welcome!
we never use more than 1%.
and yes probably the issue!!
Unless you want to isolate a membrane protein it's an overkill and if you want to isolate a membrane protein there are much better detergents yet not necessarily more compatible with common protein quantification kits.
so reduce the detergent to 1% and you want to add a protease inhibitor so your protein doesn't get chopped up. rest of ingredients of lysis buffer sounds ok.
If looking for phosphorylation of signaling proteins add a phosphatase inhibitor too...they have broad band ones available commercially (try pierce or Roche)
And lyse cells on ICE for 10 min or less.. I do 5 min and scrape and that's fine!
Then cortex in tube and centrifuge to get rid of debris and DNA and freeze or use instantly.
At RT you just give proteases a chance to chop your protein of interest to shreds.