For endogenous IP you can not use Flag beads. Flag tag beads are used for IP of transfected protein having FLAG tag. Take endogenous lysate varying from 500ug to 1mg. preclear with Ag beads. Divide the fraction in two parts. In one part add control IgG and in other part add your antibody. Pull down should be done in IP rotor at 4 degree for overnight. Next day add 20-30 ul of beads and keep on IP rotor at 4 degree for 1-4 hours. centrifuge at 1000 rpm for 3 mins. discard supernatant, wash the beads with IP buffer. elute with 2x sds dye. run on gel. Follow general western blot protocol.

Sorry u did not mentiin.... Yes i know that i can not use flag tagged beads for endo .. but i have samples with flag tagged protein. 

I *

Thanks a lot for your answer. 

Thats exactly what i did. 

Did you check my blot ? 

Can you tell me what is the efficient method to detect my protein on blot afte

Yes I did check your blot. Chemiluminescence  method will work best. Use ECL plus kit from thermo for blot development.

Dear Aftab,

Yes i used the same kit.

but i see all bands as i see with Odysee.

thanks

Is it possible that ponceau staining is interferring ?

Shall i use Silver staning instead and do Mass spec.?

Generally for IP we do not do Ponceau, it can be a reason. But in input you will get band irrespective of ponceau. Silver staining is again tricky for mass spec analysis. If you want to do mass spec do 3-4 IP and pool them and run on the gel. Stain with coomassei, cut the band and go fro mass spec.