0.1% non-ionic detergent is quite low for lysis buffer. I would recommend 1.0% to improve lysis efficiency. Be advised that lysis efficiency depends in part on the mass ratio of total detergent to total membrane mass. Too little detergent and/or too many cells will inhibit lysis when detergent amount is limiting (which I suspect in your case where total volume is quite small, and detergent concentration is low).

In addition, you are correct in your inference that you will not disrupt nuclei with this lysis procedure. Nuclear lysis as well as disruption of cytoskeletal complexes in the cytosol will require harsher conditions, such as inclusion of SDS and/or deoxycholate.

0.1% non-ionic detergent is quite low for lysis buffer. I would recommend 1.0% to improve lysis efficiency. Be advised that lysis efficiency depends in part on the mass ratio of total detergent to total membrane mass. Too little detergent and/or too many cells will inhibit lysis when detergent amount is limiting (which I suspect in your case where total volume is quite small, and detergent concentration is low).

In addition, you are correct in your inference that you will not disrupt nuclei with this lysis procedure. Nuclear lysis as well as disruption of cytoskeletal complexes in the cytosol will require harsher conditions, such as inclusion of SDS and/or deoxycholate.

Yes your question has the answer; RIPA(has SDS,cholate and NP-40) is the strong lysis buffer to get so called whole cell lysate; NP-40, with 0.1%, you get mainly the soluble protein fractions of cytoplasm. You can try different buffer in parallel with the same cell and choose the best one you needed (you dont have to strictly follow your LAB buffer/any other's..make your own!). cheers.

I agree with Daniel and Ananda. Normally, one should optimize extraction buffer for the protein he is interested in. In your case, you should also re-solubilize the pellet and assay for your protein by WB to check for the efficiency of the extraction. I would expect 5 mM EDTA to break nuclei but I am not sure about your volume. Is it a typo error? 50 ul is quite small for 60 mm dish. You need 3-5 volumes of buffer per volume of packed cells in most extraction buffers.

Also, take into account that 150 mM is not sufficient to extract most nuclear proteins. People use around 400-450, but again it depends on your protein. Some might be salt-resistant; hence it is good to include ionic detergent.

In summary, if you are only interested in performing WB, then forget about making cell extract. Just count your cells and lyse them in 2x SDS buffer or even 4X, shear the DNA, boil or better incubate a 70-80 C for 10 min, centrifuge and load different volumes (cell number)…

Thanks guys, i'll make the RIPA buffer and our lab buffer (now with 1% NP40-igepal) and check nuclear loading control (lamin etc)

@ Abdelhalim Boukaba

actually we detach the cell with 2.5% EDTA from plate, wash with pbs, and pellet the cell. to the pellet from 60mm dish, we use ~50-60ul lysis buffer

Hi, Supratim, I agree with previous comments. There is one more reason: may be low protein level were related with smal buffer volume, which can not extract proper. You have to use ratio between PSV (packed cell volume) at least 1:10. Good luck!

Hi, Supratim, please, also take into account that treatment with 2,5% EDTA and washing with PBS may alter protein abundance. Of course, it is dependent from your protein of interest. So, I would suggest to avoid such treatments.

Do not forget about what kind of cells are you dealing with. Once I used PBS to wash endothelial cells with low protein content. Later I realized that PBS without Ca/Mg can unplugg my endothelial cells which led to the low protein yield. NP-40 may be not strong enough for your cells but I had no problem before using them as long as I allow the cells to sit on ice with lysis buffer long enough. I also use RIPA buffer which sometimes is too strong and I can see sticky DNA at the bottom of my tube. When I falied to avoid it the sample will for wedge on the electrophoresis.

Just for completeness, make sure your BCA standards are prepared in the same buffer, and that this is not a simple issue with your buffer interfering with the BCA assay.

I would definitely also check that your assay buffer is not causing an issue, I know of colleagues who also have had problems caused by RIPA buffer interfering with the BCA reagent.