help me ! dying about my western blot - (Jul/25/2006 )

Dear all.
my western was great. but since couple of months ago my western showing strange,always have lots small dots on blot and could not see the band. sometime the band place show dots too. could not figure out what is the problem at all. help help. I am really dying on it. very very sad. anyone has this bad experience? help.


Thank you all very much.

Dodgy acrylamide by the looks of it. It can give nasty results when it goes off.

Could also be some incomplete transfer. I'd buy new acrylamide stocks and reagents.

Thank you for replying. I use comercial gels, so I guess should be ok. and for transfer I could see the marker transfer completely. really drive me crazy

then it's either the sample loading buffer (laemmli buffer), or the running buffer.
Did you use a new batch?
Is it worth to prepare again fresh solutions?

even the ladder is not correct...
I suspect something wrong in laemmli, or buffers...
Btw, there is a "best before" date on gels too... did you check it?
i think that it would worth to do your own gel.

I think your gel may have expired!!

they're not all gels from the same lot, are they?

only other thing, how's your detection reagent? is it clumpy at all? that stuff usually lasts well beyond the expiration date, but it can still go bad

hi kathy,
for me those looks like, they r coming from ur film. i also faced this problem, but not so intensed.
suggestions
1-wash membrane in high volumes of PBS-T (50mlX10min)
2-use freshly opened x-ray film and see whether u can get rid of it or no!
3-make sure that ur x-ray cassette is not contaminated with some fluroscent agents (put xray film alone in cassette before keeping the membrane n develope it)

i hope u got wat i m trying explain u!!
gud luk
payeli

Thank you you guys all very much. The problem is people in the lab still has good blots, so it rules out ECL problem and X-ray films problem. and I think it is nothing to do with the samples I load, because I tried the old samples where I run great blots.
I think of everything I could think, just no no idea. cry

do you share the running buffer with others?
maybe this could be the answer (maybe you made a mistake while preparing your buffer like forgetting the SDS... this can happen to everybody)
?