I would like to collect PBMCs from patients to later extract protein and do western blotting eventually. I wonder if there is a way to preserve the protein or the cells (in the field), so my samples won't ruin with time as I'm supposed to do a flow cytometry protocol on other samples in the same day.
I tried to use RNAlater from Life Technologies, as they said I can preserve RNA or protein in it. I put the cells there with 5x the volume of RNAlater, left them in at 4C for 3 days to test. Then I extract the protein and there was no much protein which was confirmed with the western blotting. I got no GAPDH bands.
Hi,
try pelleting the PBMC. Remove supernatant nad place cells inot the -20 then -80 degree freezer for later extraction.
Hi,
try pelleting the PBMC. Remove supernatant nad place cells inot the -20 then -80 degree freezer for later extraction.
You can pellet PBMCs and then lyse cells either by a commercial lysis buffer or a home made one.
I use this one
1x PBS w/o
1%triton x-100
1mM PMSF (add in the end always fresh)
1mM NEM
100ul coctail protease inhibitors (Sigma P8340)
1mM aprotinin
Use 1ml per 10^7 cells
incubate 30-60min in 4oC
Centrifuge in 4oC in 11500rpm for 15 min
Collect supernatent, store in -20oC
Hope this will work for you too
Cheers
Dear sofia,
Did you find any technique to preserve your PBMCs for later performing western blotting?
Please provide this information, since i am also stuck with this question.
Thank you.
Hi Santhosh,
Unfortunately, I did not find a way to preserve the PBMC intact, but instead I made the process shorter by collected the blood using BD Vacutainer®CPT™ Tubes according to manufacturer’s instructions. After PBMCS extraction and washing, I lysed the protein using Sigma Mammalian Cell Lysis kit (MCL-1) (Sigma Aldrich, St. Luis, USA) and assayed the protein using Bio-Rad DC Protein Assay kit.
The other thing you can do,which I also tried, is to collect the washed cells in an Eppendorf tube and remove any excess fluid around them as the water crystal will scratch and lyse the cells, then deep freeze them immediately for later process. However, you may loose some of the protein.
Hope this will help!
Sorry, it is not an answer but a question !
How many PBMC are necessary for a correct protein extraction in view of a western blotting ? (I put around 20 µg total proteins by gel track).
Thanks a lot!
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