On 19 Jul., 22:47, riccardo senter <ricc_wpa... from yahoo.it> wrote:
> hi!
> i'm a new entry.
Welcome!
> i need to extract proteins from PBMC in order to perform a western blot and a functional assay of iNOS, which is a cytosolic homodimeric protein.
> which is the best extraction protocol?
Basically, any buffer without SDS should do it. If in doubt, compare
all protocols / buffers you may get hold of with a functional assay.
My preferred start (without having checked any literature) would be
0.1% Triton X 100, Tween 20 or NP40 in PBS with protease inhibitors,
cold, with a rotor-stator homogenizer. Detergent would be just for
efficient lysis of cell walls, as iNOS is supposed to be cytosolic,
and might be omitted if they should . For SDS-PAGE, mix aliquots with
several times concentrated Laemmli style buffer and freeze. Check
protein content with some other aliquot, then adjust the amount of
protein in PAGE by sample volume.
>> Another question: can i use PBMC pellets that have been storing for several years at -20°?
Difficult to say. I would doubt that any phosphorylation signal (if
still present at all) is reliable. For protein integrity at all,
compare with fresh samples.
Good luck and best regards,
Wo
