Immunoprecipitation troubleshooting

Immunoprecipitation troubleshooting guide

Problem Cause Solution
1. High background a. Non-specific binding to agarose beads/antibodies./td> a. Add a pre-clearing stage to prevent non-specific binding.
b. Protein aggregates that are not detergent soluble. b. Extract the supernatant immediately after centrifugations.
c. Too much antibody used can leads to non-specific binding. c. Check the recommended amount of antibody..
d. Inadequate washing. d. Wash well when relevant and use more stringent washes. As well as increasing the amount of washes.
e. Non-specific proteins are binding with the beads. e. Make sure beads are pre-blocked enough in 1% BSA in PBS.
e. Frozen cells were used. e. Try to use fresh material but if not use frozen lysates as opposed to frozen cells.
2. Specific antigen is not being detected a. The antibody used is not suitable. a. Try using a different antibody for example polyclonal antibodies may work better than monoclonal ones.
b. Inappropriate beads used so do not bind antibodies. b. Check the type of beads that should be used for the antibody isotype.
c. Washes are too harsh meaning antibodies were stripped from agarose beads. c. Reduce detergent and salt, alternatively use a different detergent and reduce the number of washes.
d. Antigen of interest is not present or there is a low level. d. Ensure the sample is appropriate and, if there is a low level, increase the amount of lysate used.
e. Too many proteins competing in the mixture. e. Before adding the antibody make sure to centrifuge to remove insoluble proteins.
f. Antibody concentration too low. f. Titrate antibodies to find appropriate amount required.
g. Incubation times not sufficient. g. Increase incubation times (overnight at 4oC).