Hi everybody,
I just start to work on coIP. I am studying the interaction between a small menbrane protein A (17kd), and a big menbrane protein B (75kd). I precipitate B, and detect A by WB. I found the control (normal IgG+beads) can be detect protein A. Even I use beads as control, the band is still clear. I doubt the beads can trap small hydrophobic protein.
The beads is produced by Santa Cruz.

Should I use a stringent condition to wash beads? Or I should change another beads.

Thanks!

5X bed volume washing with your buffer + detergent (use the detergent concentration you used for protein solubilization) may work

Welcome to the difficult erea of IP :-)
There are alot of variable to change in order to get the minimum back ground with maximum IP.
As said before me try to use more washings but you may loose the protein that you are looking for.
I would start with different beads. My self I prefer GE 4-Fast flow G-beads. for my IP and CO-IP.
antother way is to try other ab's maybe you can get a much higher amount of protein A percipitated (increase you IP efficiency) so the amount of back ground regarding to the true signal will be very loe.
Guy

Hi qinglongyanyuedao & guy,
Thanks for your answers!
Yes, I think I should change the beads.
Do you use spin columns, or Ep tubes?
Can the GE beads be reused? How many times can they be reused?
How many dishes of cell do you use? And how many purified antibody do you add each trial?

Thanks!

Hi,
I am using alot of cells as I am doing CO-IP studies, For regular IP I use 10CM dish. All depends upon the expression rate of your protein.
As per the reuse of the beads, that depends upong the way you elute. If you use either acidic or bacic elution procedures you can reuse the beads.
I would not use them more then twice
I use a combination of two Ab's one is Anti HA and the other is against my protein.
You should use the same host when using two different Ab's,
Guy

Thank you!
You misunderstood my words. I means when you are doing IP, how many gram Ab you add to your beads?
Have you compared these two situations:
1 first add Ab to beads, incubate; and then add the cell lysate to the mixture
2 first add Ab to the cell lysate, and then add beads
Which procedure is better?
Thanks!

Hi ,
First you must bare in mind that evry protein have got different "preferences" :-)
For my protein a 170kDa membrane channal I tryed both cases and for me incubating the lysate with beads while rocking for 1hr at 4Deg is better then first incubating the Ab with beads. But there are some Ab that are preferebly need to be bound to the beads first.

I am using 1mg of Ab in combination with another Ab.

Thanks!

Happy I could help you,
In anycasse that you still got problems write.
Guy