DICER Western Blot - (May/08/2006 )
Has anyone here tried to detect DICER in a western blot?
I have tried several antibodies, one from imgenex (rabbit anti-mouse) and one monoclonal from Abcam. Both are not producing good results.
i tend to detect drosha and better results are if i separate nuclear proteins from cytoplasmic proteins.
I wonder if my lysis buffer could be a problem; it only has Tris pH = 7.4, 0.5% Na deoxycholate and 0.1% Triton-X. Maybe I should try a different buffer?
well i don't use strickly this buffer, but i would prefer a separation nuceleus/cytoplasm, in order to increase the relative ratio of dicer in the extracts. i assume your procedure allows a full cell extract, and maybe that's a negative point for your exp.
In my protocol I lyse the cells using the above buffer for 10min at 4 degrees and then spin the cells and remove the nuclei by replacing the supernatant in another tube (nucleus stay attached to the tube).
The company that makes the antibody that I am currently using (Imgenex) uses ovary cell lysate as a positive control. Their lysis buffer is as follows:
10 mM Tris
130 mM NaCl
10 mM NaF
10 mM sodium phosphate
10 mM sodium pyrophosphate
1% Triton X
1 mM PIC and PMSF
What lysis buffer do you use and what is your protocol for separating cytoplasm and nuclei fraction?
i gave the protocols.... here