problem in western blotting, picture attached - spots in xray film but no bands (Apr/21/2010 )

Hello all,

I did a western yesterday. and instead i found these spots instead of bands. Can anybody tell me what could have gone wrong. blocking - milk in tbs-t.
http://img535.imageshack.us/img535/1290/20...11119025123.jpg
please zoom out to make it clearer. thatäs the scan i have

many thanks in advance

kedar

Looks like you either added antibody directly to the membrane so that it all bound in one spot (dilute them before adding), or the substrate wasn't mixed properly.

bob1 on Apr 21 2010, 04:52 PM said:

Hi Bob1,

thanks for the reply. i dilute the primary- 1:200 and seconbdary antibody 1:10000 in blocking buffer(5% milk in tbst).

substrate- not mixed.--- may be...but you can imagine the odds here, the substrate is from biorad-immune star HRP luminol enhancer, i just need to mix it with buffer and submerge the blot ...which i did...

any more ideas?

thanks'kedar

kedar on Apr 21 2010, 09:28 AM said:

contamination of film cassette or the plastic cover?
did you stain with Ponceau S to show proper transfer of the polypeptides to the blot?
the best is to strip the blot and repeat it;