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High background in immunoprecipitation (IP)

Can be caused by specific or non-specific binding.

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.