the weak signal in the middle bane,blur and high background - Western blot : caspase3 (Nov/13/2007 )

hello
I'm doing some experiment about caspase3.

In the picture, first one is B-actin it looks ok for me but caspase3 protein, the bane is quite blur and in the middle bane is quite weaker than the border bane and background is quite black.

As you see the actin, it looks sharp and straight, so I don't think it would be something wrong with my loaded samples or maybe not :\


-separated 12% resolving gel about 1.5 hr
-my protein concentration about 1 ug/ul and I loaded about 40 ug each well.

-blocking in 5% free fat milk in TBST for 2 hr and following with 3 time washing with TBST
-I use 1-ab rabbit poloclonal anti active canpase3 1:300 (chemicon international) in 3% free fat milk, TBST for 4 C overnight (about 30 hr) then waching 3 time
- 2-ab donkey anti rabbit in 3% free fat milk 1 hr (santacruz) then washing 3 time
both antibody are new one.

then I use ECL plus for detecting signal

If any one have suggestion I'd really appreciate.
thank you in advance
Tai

How do you expose your films? Sometimes I see this pattern like the right one, when I only put my handy on the film and there is some distance between membrane and x-ray film. You should put your membranes and films in cassettes to get a could pressure, perhaps this might get you sharper bands.

I take a picture by Fuji CCD camera with chemiluminescense light according of ECL plus kit , Amersham and I take a picture directly on the PVDF membrane. I didn't use x-ray film.

Today I just increase concentration to my samples. I'm not sure if it'd work. will see tomorrow

Thank you biomaus for your suggestion