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!
You do need to lyse the cells properly, sonication should do this reasonably effectively I would have thought, though duration and frequency play a big part in this.
10% triton is a massive amount of detergent, we use 0.1-1% depending on the buffer and they work fine for a 30 min lysis. The triton will be interfering with your BCA.