I am getting strong bands for both bait and prey protein in starting material and flow through, only weak band in elution lane for bait protein and none for prey protein. I am using Pierce Co-IP kit from Thermo Scientific.
I will appreciate if some expert can give input into this query. Thanks.
Since you have only a weak band or no bands in the flow through and Elution, it seems to me that you are not effectively eluting your protein, especially since you see them in the starting material. I would take the beads, from the co-IP before Elution, and run that on a gel, to see if you pull down the protein of interest. If you do, then you know that the Elution is not working.
The information is not really clear but two possible probabilities include: does the procedure require that you autozero using a specific type of blank? Secondly, does the kit contain enzyme that could carry out breakdown during assay? Also check compatibilities between kit materials and samples for possible adsorption properties. I humbly suggest that you be a little more detailed to enable comprehension by wider audience. God bless
Hi there,
4 possibilities:
unefficient antibody/beads crosslinking
unefficient pull down (check antibody)
unefficient elution (check elution condition)
weak interaction destroyed during the CoIP (try to find condition suitable for stable interaction)
Since you have only a weak band or no bands in the flow through and Elution, it seems to me that you are not effectively eluting your protein, especially since you see them in the starting material. I would take the beads, from the co-IP before Elution, and run that on a gel, to see if you pull down the protein of interest. If you do, then you know that the Elution is not working.
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