Hi Nirmal,

have you used Protein A/G or L for the Co-IP? In this case maybe you also precipitated endogenous IgG from your plasma sample. Then you can maybe try an pre-clearing with your beads or biotinylate your capture ab and switch to Streptavidin or Neutravidin beads.

Good luck!

Carsten

Do you have good protocol to pre clear beads?

Hi Nirmal,

it is difficult to estimate how much endogenous IgGs are in your samples. I would try to use at least1x-2x the amount of beads you use for your IP, in the same buffer under the same conditions. I have used 10 µl of beads for my normal IP, so I have used for pre-clearing 20 µl beads, but you have to try.

Good luck,

Carsten

Hi Nirmal,

I have the same problem. Have you resolve this problem?

hi

I use Pierce co-IP kit for this purpose. It might help u. so I immoblize the ab on to A/G beads and then i perform the Experiment.

I see no Background. WB is specific and clean.

We got it developed in house by eurogentec. We have Monoclonal ab working nicely. Got Point about These resins are that they are reuseable so you dont really end up wasting ab

Sneha,

Thank you for your reply! 

Hi Carsten:

Thank you for the suggestion. I am using Dynabeads® Striptreptavidin and biotinylated antibody and would like to elute the protein of interest. Do you have any suggestion on how much plasma to begin with? Thank you.

Hi Fereshteh,

difficult to give a recommendation, depends on the abundance of your target protein. I have used 400 µl of plasma for an IP. But before use I have diluted it 1:1 with PBS-CM (PBS with 0.5 mM CaCl2 and 2 mM MgCl2).

Good luck,

Carsten