Dear Gi!

Maybe you meant extract total protein from PBMC for my western blot analysis

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

Daniela Bruennert added an answer August 26, 2016

Well Felix, unlike for tissue, for PBMCs it is not required to grind the cells, but you should wash them anyway 2x with 1x ice cold PBS (usually for 5 min, 500g) before lysing them with the respective lysis buffer. Depending on your protein of interest, the lysis buffer might be TRIS based (and even here you might have to check if a concentration of 10 mM, 25 mM or 50 mM works best), or HEPES based. Sometimes, a bit of CHAPS inside helps also. For primary cells, Thermo Fisher even offers a specific buffer called M-PER Mammalian Protein Extraction Reagent. And depending on your protocol, lysis might take anything from 10min to 1 hour, I have seen a lot of different protocols. For sonification of cells, 5 min is too much, normally we use 3-5 pulses with 20% intensity and something around 10-20 sec.

Mary Cristine Charlesworth added an answer August 26, 2016

if you only want to do western and not retain functional activity of proteins in the lysate, the easy way is to wash the cells as suggested, then add a small volume (100ul) of 0.1% SDS, 50mM Tris, pH 8.0. You can also add Bensonaze or other nuclease to digest up the DNA. 0.5 ul is sufficient. Add this cold buffer to the cell pellet, vortex at max speed for 20 seconds, let sit on ice 5 min. Repeat twice. Heat at 85C for 10 min. Done. Do not centrifuge - the lysate will be a little cloudy or milky in appearance which is OK. You can do a BCA assay (1:5 dil in micro plate assay) for protein determination in presence of SDS (no reducing agent). You should get a yield from 1 million PBMC of ~40-50 ug protein. You can add reducing agent in SDS loading buffer to an aliquot of the sample and heat another few minutes if you want to reduce proteins before loading on gel.  If you normalize what you load on the gel by cell number and not protein, you can just add SDS-PAGE buffer with bME or DTT directly to the washed pellet. Vortex and heat as above. Everything will solubilize and then just load the same volume of sample to all the lanes. We do this all the time with cell pellets and it works great IF you only want to run gels on the lysate, because everything will be denatured of course by the SDS.

Mohamad Fairus Abdul Kadir added an answer March 28, 2017

Hi Kamariah, If you are still finding difficulties in isolating protein for PBMC, this could be helpful. I have used 8 million PBMC with approx 50-60ul of RIPA buffer which included protease inhibitors and phosphatase inhibitors. BCA protein determination gave me approximately 600-800 ug/ml total protein. So, if you have lesser cells i suggest you to reduce the ripa buffer volumes to concentrate the total protein. Hope this helps.

To assess for contamination of serum proteins in ficoll- isolated pbmc using WB, I would test for the presence of human serum albumin. There are a number of anti- hsa antibodies that can be used to detect hsa via WB. Cell signaling sells one.

You are welcome Gi ; )