Western Blot problem! - (May/15/2008 )

Hi friends!

I'm having a lot of problems with my protein in western blot!
With chemiluminescence I have a lot of background and I cant see the band! And I can only see the band when I stain the membrane with dab!
Here is my protocol:
My recombinant protein has 20KDa.
I transfer for nitrocellulose (100 ng of protein) in semi dry system for 20 minutes, block with 5% milk, 2 hours of primary antibody (1:1000), wash 3 times of 5 minutes with PBS-Tween (0.1%), 1 hour of protein A peroxidase (1:4000), wash 3 times of 5 minutes with PBS-Tween (0.1%) and I use ECL of millipore for detection.

Another question: Is dab quantitative?

Can anyone help me, please?

Thanks!
Teka

Hi:)
Your background problem could be due to a couple of things. Have you done other Westerns and they're OK, and it's only this one that's problematic?
Are you wiping excess ECL off? (I normally would wet my membranes in a shallow dish, then lift up the membrane by a corner using tweezers, and let excess drip off, then touch a small corner onto the dish edge, again to dry it off a bit. Otherwise if you're dripping your ECL onto the membrane, then you can just tilt it and dab at the sides with a tissue)
Depending on what your antibody is, milk may not be the best thing to block with, it could actually be picking up yout antibody, rather than blocking the sites. You can try using a solution of FBS or horse serum, or a protein digest eg Polypep in PBS-T, or whatever you normally would use to make the milk in.
Try varying the length of time you expose the film for, if this is possible, sometimes 10 seconds literally is enough.
Decrease your 2AB concentration?
This is all I can think of so far.

Hi! Thanks for your answer! This problem only happens with this antibody, others antibodies my western is ok!
I always wip the ECL excess! But I'll try your method!
And I'm exposing the film 10 sec! I'm increasing the dilution of the primary antibody, it´s seems that its getting better, but i still have background!
What do you think?
Teka