weak signal! ! ! !

[moganti]

Hello everyone,

I have been doing the Western blot since 3 months.I have loaded 30ug of protein samples and first checked with alpha tubulin if I loaded the samples correctly and then added the desired antibody (cleaved caspase-3) .....I got the signals with controls but not with the antibody that I want.. and the signals are very low and invisible ...........the reccommended dilution is 1:1000 which I have added...Can anyone please give me the solution to get the results....

becoz I didnt find my signals .....

I added 5%milk/TBS-T
Primary antibody is cleaved caspase-3(1:1000)
Secpndary antibody is antirabbit (1:10,000)

[admin]

Hi Moganti,
can you post an image of your gels/blots?

May be several issues:
- insufficient blocking during primary or secondary incubation to minimize non-specific binding
- poor antibody (has anyone ever got good bands with the antibody?)
- poor sample quality (are you looking for a phosphorylated protein or modified protein or a protein that is degraded quickly?)

just a few questions

cheers

[Anastasia_S]

Hi Moganti,
It may be that because your cleaved caspase-3 concentration in samples is so small, you'll have to overload your gel (add much more than 30ug protein/well) to get a detectable signal

[A.A]

Hi Moganti

I would

1/Titrate the antibody (use serial dilution start from 1: 100)
2/ increase the protein concentration
3/ use +ve control if possible

All the best

[Tfal]

How abundant is your target protein? If it's concentration is too low, simply adding more 'total' protein might not be sufficient. You might have to narrow your fraction.