Western blot has alot of spots - (Mar/09/2009 )

I have alot of spots on my western blot (looks like a starry sky) I know it cant be because of my anti mouse HRP conjugating cuz my beta actin control (which also uses the same anti mouse ) does not have the problem. Thinking it might be because my membrane might have been dirty prior to using it for transfer, i stripped the beta actin control membrane, inucubated it for a shorter secondary antibody incubation time (1hr; previously it was 1hr 30 minutes) but this time I have no bands at all, but the starry sky is still there. I have stripped again, now i am incubating with a lower Ab concetration of 1:10,000 instead of previous 1:1000. Havent developed yet.


Have any of you guys had the same problem?

I remember that someone has such problem. He solved the problem by preparing new SDS solution.

little mouse on Mar 9 2009, 12:41 AM said:

i used the same SDS for my beta actin control but there is no problem there. I will prepare new sds anyway. thanka

any possibility that they might be bubbles trapped between gel and the membrane during electroblotting?

If I understand well, you perform WB anti-beta actin : all is fine,

if you strip this membrane and blot with your antibody of interest, you have a starry sky.
The same with a membrane freshly transferred and blotted with your antibody of interest.

if yes, don't lose time with preparing new SDS, it is not responsable for your problems.

what do you use as blocking reagent? milk?
do you blot against phospho-protein? biotinylated protein?

I have similar problem but only with 2 antibodies for caspase-10 and GRP78. for many other antibodies (over 25) I do not have that problem. It seems to be related to the the antobody source.