I'm currently conducting research with human subjects and would like to analyze human neutrophils by Western Blot. We get 1-2 patients a week -- would it be possible to flash freeze the neutrophils and lyse them later, or is it better to prepare Western samples using fresh neutrophils?
flash freezing the neutrophils and later on lysing them will not be a problem as per me. there must not be great difference between these two categories. The best approach that i can suggest would be sacrificing a few samples and do a comparative check for these two approaches.
wish u all the success.
Cheers!!!!
In my experience it is better to lyse and prepare the samples immediately, rather than store them for processing at a later date. Once you have prepared the samples, you can then store them at -80 for a while (have tried up to three months) before running the WB if you really need to.
Thank you! do you have any recommendations for a lysis protocol?
I have always preferred an aggressive approach as I have found them to be somewhat resistant to lysing and chock full of proteases. A good description can be found in my old JLB paper. I have tried syringing instead of the alternate freeze/thaw described below but believe the syringing ruptures the nuclease and reduces the quality of the RNA.
Good luck!
"lyse in 50 mM HEPES, pH 7.6, 75 mM NaCl, 0.25% deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 1 M NaF, 5 mM MgCl2 with protease inhibitor cocktail (P-8340, Sigma Chemical Co.), phosphatase-inhibitor cocktail
(P-5725, Sigma Chemical Co.), 2 mM phenylmethylsulfonyl fluoride, 100
M sodium orthovanadate, 900 M benzamidine, and 1 mM phenantroline.
Samples were subjected to three alternating freeze/thaw cycles, and the debris
pelleted."
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