Problem 1：High background
Possible Source	Test or Action
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 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
Antibody used contains antibodies that are    not specific enough	Use an affinity purified antibody, preferably    pre-adsorbed.
Too much antibody used leading to    non-specific binding	Check the recommended amount of antibody    suggested. Try using less antibody
Too many cells/too much protein in lysate    leading to a lot of non-specificproteins in eluate	Reduce the number of cells/lysate used. We    recommend using 10-500 µg cell lysate..
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.
Antigen degrading during immunoprecipitation	Ensure fresh protease inhibitors are added    when sample is lysed.

 

Problem 2：High amount of antibody eluting
Possible Source	Test or Action
Too much antibody eluting with the target    protein	Try reducing the amount of antibody.    Crosslinking the antibody to the beads before the immunoprecipitation and eluting using a    gentle glycine buffer gradient should significantly reduce the    amount of antibody eluted.

 

Problem 3：No eluted target protein detected
Possible Source	Test or Action
Target protein not expressed    in sample used/low level of target proteinexpression in sample used	Check the expression profile of the target    protein to ensure it will be expressed in the cells of your samples.    If there is low level of target protein expression, increase the amount of    lysate used. However, this may result in increased non-specific binding so it    would be advisable to pre-clear the lysate before commencing with the IP    procedure
Insufficient antibody for capture of the target protein	Check that the recommended amount of antibody is being    used. The concentration of antibody may require increasing for optimization    of results.
Target protein has not eluted from the beads	Ensure    you are using the correct elution buffer and that it is at the correct    strength and pH for elution of the protein.
Antibody has not bound to immunoadsorbant beads	Ensure you are using the correct beads for the antibody    isotype used.
Incorrect lysis buffer used	Check    datasheet to see if the antibody detects denatured or native protein and    ensure the correct lysis buffer is used.

 

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