Possible Causes

What You Can Do?

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Insufficient antibody

Check the recommended amount of antibody as indicated in the datasheet.  Titrate and optimize the optimal antibody amount used per IP experiment.

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Washes too stringent

Reduce the number of washes. Reduce salt concentration in the wash buffer.

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Incorrect Protein A/G/L used

Make sure that the Protein A/G/L beads are capable of binding to the antibody subclass being used.

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Target protein not present in the
sample used

Make sure that the target protein is expressed at a relatively high level in the sample used by including an Input sample in the WB.

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Incorrect Lysis buffer used

Make sure that the lysis buffer used is not over-denaturing and destroy the native conformation of the target proteins.

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Antibody not capable of
immunoprecipitation

Try a different antibody. Try polyclonal antibody if monoclonal antibody does not work well.

Possible Causes

What You Can Do?

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Secondary antibody rcognizes heavy / light chain denatured from primary 
antibody

Use secondary antibodies which only recognizes native form of IgG for immunoblotting.

Possible Causes

What You Can Do?

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Inadequate washing

Use a more stringent washing buffer. Try to  use a high salt washing buffer or add 0.2% SDS or 1% Tween20 to washing buffer. Increase the number of washes.

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Concentration of antibodies too high

Check the recommended amount of antibody as indicated in the datasheet.  Titrate and optimize the optimal antibody amount used per IP experiment.

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Non specific binding to Protein A,G or L

Pre-block beads with BSA. Incubate beads with 2%BSA in PBS for 1 hour wash in PBS before use.

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Non specific binding to agarose beads

Include a pre-clear step by incubating lysate with Protein A/G/L agarose beads.

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Antibody not specific enough

Use affinity purified and pre-absored antibody for IP experiments.

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Sample degradation

Add adequate protease inhibitors and phosphatase inhibitors throughout sample preparation and Ip steps.