Dear Heba, 

If you have a low number of cells you might not obtain any visible pellet after centrifugation. RIPA contains enough detergents to solubilize membrane proteins so in supernatant you will have most of the soluble proteins and many of the proteins attached to membranes. I do not think that you will have problems to detect a protein by western blot but you can  collect more (in case it is possible) cells to obtain a higher  protein concentration after lysis

Among most of the lysis buffers , ripa gave the least pellet. And of course with great concentrations. U should anyway quantify ur proteins or run a trial sds gel before u start western. When I take cells from 35mm dishes I hardly get any pellet. But when I take a 150 mm dish I do see . If it's true u dint have the protein , give us more details like how many cells. What type of cells .. ratio of cell to lysis buffer etc. 

Dear Heba,

Just to understand: do you collect the cells first and then you add the RIPA?

What I know is that our RIPA buffer contains NP-40, a detergent that disturbs almost all membranes, except the Nuclei. 

So, if you want to collect the nuclei after the lysis, I agree with Asien. It could be that you have too little amount of cells. Usually nuclei should pellet at the speed you are using. Lower speeds work as well but I do not remember the ideal speeds at the moment.

But if you want to have all the proteins from the cells, you will need to collect them first by using PBS 2mM EDTA,  for example,  and centrifuge at max. 500g (higher g forces can cause that cells explode) for 10 min at 4°C and then lyse the pellet with small quantity of RIPA buffer. I use 20 microliters for a pellet of 1x10e6 amd the 25microliters of 2X SDS-loading buffer.

I hope it helps you.

Dear Heba,

I think that Luisa's answer is the best one of the three.   I would follow her advice.

I got the same problem. my fibroblast cells were cultured to the 6-well plate. I added 200mcl of M-PER™ Mammalian Protein Extraction Reagent/ Protease and Phosphatase Inhibitors each well. I used the plastic crape to dislogde the cells, the centrifuged at 16000 for 20m, but no pellet. However, when determined the protein concentration, the values were ok. I have done the same protocol for HL-1 cells, by that time I got the pellets all the times.

Luisa F. Jimenez-Soto i am curious about that after you collect the cell from PBS-EDTA, you add the ripa buffer for lysis,so can you directly use this cell+RIPA mixture for SDS page or WB???