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integrin beta1 western - (Jun/04/2008 )

HEllo all,
I'm trying to do a western blot for itgb1. I extracted the protein using RIPA buffer from endothelial cells- ran 50ug of the prtein on 8% SDS gel-I checked for the transfer using ponceau (which looked perfectly fine). I blocked the memberane in 5% milk for 1hr at RT and incubated the primary antibody (from R&D systems) in 5% milk at 1:500 dilution. I did not see any band at all.
Could you please suggest any protocol or a different antibody that worked for you pls-I'll really appreciate that.
Thanks!!

-ruchi-

QUOTE (ruchi @ Jun 4 2008, 02:55 PM)
HEllo all,
I'm trying to do a western blot for itgb1. I extracted the protein using RIPA buffer from endothelial cells- ran 50ug of the prtein on 8% SDS gel-I checked for the transfer using ponceau (which looked perfectly fine). I blocked the memberane in 5% milk for 1hr at RT and incubated the primary antibody (from R&D systems) in 5% milk at 1:500 dilution. I did not see any band at all.
Could you please suggest any protocol or a different antibody that worked for you pls-I'll really appreciate that.
Thanks!!

Try one or some or all of these, in order of priority:

1. don't boil the samples if you are boiling. Itgb is a membrane protein, which don't fare well with boiling.
2. Use a control lane where you have itgb overexpression.
3. Change the membrane (nitro to pvdf, one comp to another)
4. use 3% milk for primary incubation o/n.
5. change ab
6. change ab concentration
7. This sunday go to church and confess all your sins.

-cellcounter-