This is the cells after 15 minutes shaking

M-PER Mammalian Protein Extraction Reagent, 25 ml, sufficient reagent to extract protein from approximately 2.5 g of cells .

M-PER Mammalian Protein Extraction Reagent, 250 ml, sufficient reagent to extract protein from approximately 25 g of cells .

M-PER Mammalian Protein Extraction Reagent, 1 L, sufficient reagent to extract protein from approximately 100 g of cells .

I hope that is help.

Edward Cubela we already have the 250ml reagent. I'm asking about the most appropriate volume for each 1 million cells and i attached a picture for my sample after shaking for 15 minutes and i am wondering about the floating white things if it is undissolved cells or degraded proteins.

Thank you.

In our lab, we add 1 ml of MPER reagent to the cell pellet (approximately for 3 million cells) and homogenize it thoroughly with a 26G needle 5-6 times or use a homogenizer at the minimum speed for 30 seconds. And we vortex it for 5-10 minutes and not shake.

The amount of MPER to be added may also depend on your need, if you want a relatively concentrated protein solution, use approximately 200 ul and do not add 1 X PBS with MPER as it may dilute the buffer.

Furthermore, the floating cells may be a result of your previous centrifugation speed for the cells. If you are using spinwin, spin or centrifuge at lower centrifugation to avoid clumps of cells. We too struggled with this problem initially during experimentation.

Hope this helps.

Best Regards.

For your clumping problem you may approach this thread:

https://www.researchgate.net/post/Can_anyone_help_with_cell_clumps_after_Ficoll_PBMC_extraction

Thank you so much @rohan ramesh pawar for your helpful answer. I optimized my protocol for using 200 ul per 1 million cells and it worked. Also, increasing the incubatio period was helpful.

Best.