too many bands in western blotting - (Jul/30/2004 )

the background is very low, does it means the blocking by milk is good?

but i get many bands, such unspecific binding make the result unclear. How to reduce such troublesome?

1:1000 for the first and the second antibody

thanks

my sample is the cell lysate, I can get 10 or more bands is one sample, actually there should be only one band at most.

Hi,


Western-blot can be look like Coomasie-blue staing if your overloaded your track with cell lysate.
Try low down amount of cell lysate when you loading of your SDS-PAAG. For example, make one gel and load into the first well 20 mkl of your lysate, second - 10 mkl, ... , 5 mkl, 2.5 mkl, and so on. And find optimal amount of lysate per well for your experiment.
Increasing of the primary and/or secondary antibody dilution as well can help.

Good luck!

Oleksii

Hi:
I think you should check the intensity of your target band! If it is very strong you can try to short the incubation time of Ab that will make your background much more clearly. Another possibility as you said, the blocking is not good.you have to try long time or little high temperature.
good luck!