Protocol Online logo
Top : Forum Archives: : Protein and Proteomics

Surprise!Does anyone have this kind of result when doing co-IP? - (May/30/2008 )

Hi all:
Could anyone answer this question for me? My co-IP results totally are beyond my expectation. They are even better. I was trying to use anti-EGFR to pull down EGFR and its interacting proteins. Theoretically, EGFR's interacting proteins should not be present in beads only reactions. Unfortunately, it was stuck on protein A beads. It was pulled down by protein A beads even without the presence of anti-EGFR. To get ride of backgrounds, I tried to increase the salt concentration in washing buffer. I even increased to 500 mM NaCl. It turned out that washing buffer with high salt did not wash out the background signals from protein A beads at all. It enhanced the cross-link between EGFR and its interacting proteins. How can the salt concentration increase the cross-link between two proteins? Does this happen to you before?

Phoebe

-phoebechiu-

QUOTE (phoebechiu @ May 30 2008, 12:53 AM)
Hi all:
Could anyone answer this question for me? My co-IP results totally are beyond my expectation. They are even better. I was trying to use anti-EGFR to pull down EGFR and its interacting proteins. Theoretically, EGFR's interacting proteins should not be present in beads only reactions. Unfortunately, it was stuck on protein A beads. It was pulled down by protein A beads even without the presence of anti-EGFR. To get ride of backgrounds, I tried to increase the salt concentration in washing buffer. I even increased to 500 mM NaCl. It turned out that washing buffer with high salt did not wash out the background signals from protein A beads at all. It enhanced the cross-link between EGFR and its interacting proteins. How can the salt concentration increase the cross-link between two proteins? Does this happen to you before?

Phoebe


it appears to be a hydrophobic interaction which will be strengthen in the presence of high salt; you may use an alternative to protein A such as Dynabeads...

-The Bearer-

Some proteins are "sticky".
In addition to salt concentration the concentration of
detergents is also very important. As are the number of washes.
Increase the amount of detergent you are using
and do more washes. Even try different detergents.

-mikew-

Some proteins will bind unspecifically directly to the Dynabeads Protein A. The beads have a slightly hydrophobic surface and can therefore bind hydrophobic proteins. This will be increased by high salt concentrations, but can be decreased by adding detergents, such as Tween-20 to a final concentration of up to 0.1%. The proteins are not being crosslinked to the beads, however, the hydrophobic interactions may sometimes be rather strong.

If you see no improvement with reduced salt and added detergent, you could try 'pre-clearing'. This involves removing proteins that bind unspecificall to the beads by adding 'naked' Protein G beads to your sample before doing the actual IP.

-Roald-