Hi,
I've done western blot on LV tissues and I want to prepare whole cell extract for west blot analysis using frozen gonads and uterine tissues as positive controls for my proteins of interest. As my work is heart-related, I have no expertise in sex organs. I am after a protocol for protein extraction from those tissues. Appreciate your help.
Thanks in advance
Keep on dry ice. Cut small piece and use beads method to get your lysate. Uterus might be more difficult to do and needs to be chopped by blade into small chunks. Or you may put a small piece into mortar, pour liquid N2 on, then crush it before adding lysis buffer.
Hi Ru,
Thanks for the reply. Actually, I've just tried with the uterus but haven't checked the protein conc. yet.
Briefly, using NE1 buffer, the cytoplasmic fraction has been extracted (10mM Hepes; 10mM Mgcl2; 5mM Kcl; triton 0.1% & protease inhibitor) by homogenization and spinning at 2,600 g for 10 min. at 4C.
To obtain the nuclear extract, the pellet was washed again with NE1 buffer and resuspended in NE2 buffer (glycerol 25%v/v; 20mM Hepes; 1.5mM Mgcl2; 500mM Nacl; 0.2mM EDTA & protease inhibitor). After 1hr incubation on ice with vortex every 5 min., the resuspended pellet was centrifuged at 10,600 g for 10 min.
Is that the protocol you recommend?
For testes, I couldn't find something helpful.
Thanks for your input.
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