High Background in Immunoprecipitation (IP)

1. I have cross-linked my antibody to the Immunomagnetic beads, but there are still antibodies drop off the beads during elution.

Cross-linking will never be 100%. Some antibodies are not cross-linked and may come off under acidity or alkaline elution. Perform a washing step with low pH directly after cross-linking to remove non-cross-linked antibodies. Remember to bring the pH back to normal before IP. Other methods is choosing the Sino Biological primary antibody conjugated immunomagnetic beads,and if your target protein is tagged ,you also can choose the sionbiological tag antibody conjugated IP KIT.

2. Too much antibody eluting with the target protein

Try reducing the amount of antibody. Crosslinking the antibody to the beads before the immunoprecipitation or eluting using a gentle elution buffer gradient should significantly reduce the amount of antibody eluted.

3. Secondary antibody recognizes denatured/reduced primary antibody released during the IP procedure or endogenous IgGs 

Using secondary antibodies, binding the heavy and light-chain of the primary antibody for WB detection of IP samples will always show up two bands (the heavy-chain at 50kDa and the light-chain at 25kDa).
To avoid disturbance by the antibody chains we recommend using secondary antibodies, such as specifically bind native (non-reduced) primary antibodies.