In our lab we isolate cells from a cancer tissue, now in passage 5, but when I count the cells with a hemocytometer I count about half million of total cells in a CONFLUENT T 75! I thing is few cells (sorry for my english). When I saw the cells in a microscope, they are big , could be it a explication for the poor count?
Hola. you have your membrane blocked, so it is´nt necessary re block. your second primary ab recognizes other epitopes in other proteins that will react also if you use this second primary ab in the first WB, because it seems less specific. I think that your first ab is monoclonal and the second policlonal,aren´t they? Good luck
Hi! Both ab are monoclonal (in first and second incubation), may be the concentration of antibody was too high, I would try with other type of antibody (rabbit origin) and later I tell you. Thanks! you are very kind! Sorry, you speak Spanish?
Well, if your transfer buffer is with SDS no problem (well we use the running buffer with HCl-tris , glycine and SDS), but with MeOH I donīt know, may be the proteins with MeOH lose their negative charge (contributed by loading sample buffer) and it donīt run well.
When you, finish tell me about how run the gel.
try to use the antibody for a freshly made Western blot to see if it makes really several unspecific bands; to increase specifity, you may decrease concentration of the primary antibody or developing time...
The western blot was made last Monday and concentration of the primary antibody was 1:200. Before with my labgroup made a WB with p16 antibody and worked fine. But my question is if I should re-block the membrane , certainly I take your advise and decrease concentration of the 1° antibody to rule out. Thanks! you're very kind.
I used to use PDVF membranes without stripping to reprove other antibodies with different weight and it worked, but in the last time, when I incubate the membrane (without re-blocking with milk 20%) unspecific bands appears in the revealed. What can i do? I want to see p16 protein in my membrane.
I use a sds-page gel, PDVF membrane transfered in semy-dry with transfer buffer with 20% MeOH, without SDS. I block the membrane with milk 20% (in TBS-T 0.4%) and incubate overnight, then I wash 3 times with TBS-T 0.4X, incubate 1 hour with the secondary antibody (1:5000 wash 3 times again with TBS-T and then developing with ECL (Thermo)