Weak signal in WESTERN BLOT - Western Blot problem (Jun/09/2009 )

Hello Everyone,

I had a problem with my Western Blot....I am trying to detect the caspase 3..So I used the cleaved caspase3 antibody and anti rabbit ab as primary and secondary antibodies..I found that the signal is very weak,(invisible)..Then I tried with alpha tubulin,loading was fine....
Could any help me to get the better result please..

I used 10% gel,

Blotting : The size of my gel is 8*5*5mA...SO I used 200 mA current with 400 Volts..with Nitrocellulose membrane...

and the dilution of my primary and secondary antibodies are 1:1000,1:10,000.....

Blocking:5% milk/TBS-Tfor 2 hrs

These are the conditions I used for my work..

Could any one help me to get the better results please

Thanks in Advance,
Moganti

Try titrating your primary antibody for the caspase-3, 1:1000 may be too dilute. Do you have a positive apoptosis control?