My EDTA blood samples are stored at -20 for a couple of months. How to isolate the leucocyte fraction to get proteins out of them? Also how much starting blood samples are required to get about 50-60 ug protein. TIA
See Akor-Dewu et al. (2013) as an example:
"To isolate leucocytes from frozen whole blood, a 200 µl aliquot was thawed; 50 µl was mixed with 1 ml of PBS in a microcentrifuge tube and centrifuged for 7 min at 700× g, 4°°C. The supernatant (containing lysed red cells) was discarded and the pellet suspended in 1 ml of PBS and centrifuged again for 7 min at 700× g, 4°C. The pelleted cells resuspended in PBS were used for the comet assay either at this stage or after repeated washing and centrifugation."
Article Leucocytes isolated from simply frozen whole blood can be us...
See Crosley et al. (2009) for protein reference values in whole blood fractions
https://dx.doi.org/10.1007%2Fs12263-009-0121-x
Though your yield will depend on method of lysis amongst other factors; it would be best to do a series of preliminary tests on a dispensable sample.
Murtaza S Nagree Thank you for your reply. I actually don't want to do comet but to isolate proteins for SDS-PAGE. Hence I guess will need more starting material, 50 ul is not sufficient
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