High background in immunoprecipitation (IP)

Can be caused by specific or non-specific binding.

Why is there high background?

Possible causes
Solution
Antibody used contains antibodies that are not specific enough
  • Use an affinity-purified antibody, preferably pre-adsorbed.
Antigen degrading during immunoprecipitation
  • Ensure fresh protease inhibitors are added when sample is lysed.
Carry over of proteins that are not detergent soluble
  • Remove supernatant immediately after centrifugations. This should leave insoluble proteins in the pellet. If resuspension occurs, centrifuge again.
Incomplete washing
  • Wash well at relevant stages by placing a lid on the tube and inverting several times before centrifuging.
Non-specific binding of proteins to antibody

  • If there are many proteins binding non-specifically, then try reducing the amount of sample loaded onto the beads.

  • You can also pre-clear the lysate by pre-incubating the prepared lysate with the beads before commencing with the immunoprecipitation. This should clear the lysate of any proteins that are binding non-specifically to the beads. Some researchers also use an irrelevant antibody of the same species of origin and same Ig subclass to pre-clear the lysate.

Non-specific proteins are binding to the beads

Beads are not pre-blocked enough with BSA.

  • Make sure BSA (fraction V) is fresh and incubate fresh beads for 1 hr with 1% BSA in PBS. Wash 3-4 times in PBS before using them.
Too many cells/too much protein in lysate leading to a lot of non-specific proteins in eluate
Reduce the number of cells/lysate used. We recommend using 10-500 µg cell lysate.

Too much antibody used leading to non-specific binding

Always check and follow the recommended amount of antibody suggested, however this can also be optimized to suit your experiment.

  • Try using less antibody.