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Immunoprecipitation - General Tips/Procedures and Troubleshooting

Immunoprecipitation (IP) is a method that enables the purification of a protein. An antibody for the protein of interest is incubated with a cell extract so that the antibody will bind the protein in solution. The antibody/antigen complex will then be pulled out of the sample using protein A/G-coupled agarose beads. This physically isolates the protein of interest from the rest of the sample. The sample can then be separated by SDS-PAGE for Western blot analysis.


General Tips and Procedure


Lysis buffers

The ideal lysis buffer will leave proteins in their native conformation, minimizing denaturation of antibody binding sites while at the same time releasing adequate amounts of protein from the sample for subsequent analysis. Nonionic detergents such as NP-40 and Triton X-100 are less harsh than ionic detergents such as SDS and sodium deoxycholate. Other variables that can affect the success of IP include salt concentration, divalent cation concentration, and pH.
As soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin. These events can be slowed down tremendously if samples are kept on ice or at 4°C at all times and appropriate inhibitors are added fresh to the lysis buffer. If not using a cocktail, two of the most commonly used protease inhibitors for IP are PMSF (50 μg/ml) and aprotinin (1 μg/ml).

Preparation of lysates

Prepare lysates from cell culture or tissues according to appropriate protocols.

Pre-clearing the lysates

Pre-clearing the lysate can help reduce non-specific binding of proteins to agarose or sepharose beads. Preclearing with an irrelevant antibody or serum will remove proteins that bind immunoglobulins non-specifically. The end result will be a lowering of background and an improved signal-to-noise ratio. However, if the final detection of the protein is by immunoblotting, pre-clearing may not be necessary, unless a contaminating protein is interfering with visualization of the protein of interest. It is important to make sure that as much of the normal serum as possible is removed as this will compete with the specific antibody for antigen.

Immunoprecipitation

It is highly recommended  that researchers review the methodology and variations in immunoprecipitation protocols (see also references at the end of this section).


Immunoprecipitation - Troubleshooting Tips


High background

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 the BSA (fraction V) is fresh and incubate fresh beads 1 hour 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-absorbed.

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-specific proteins in eluate
Reduce the number of cells/lysate used.

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.

Antigen degrading during immunoprecipitation
Ensure fresh protease inhibitors are added when sample is lysed.


High amount of antibody eluting

Too much antibody eluting with the target protein
Try reducing the amount of antibody. Cross-linking the antibody to the beads before the immunoprecipitation and eluting using a gentle glycine buffer gradient should significantly reduce the amount of antibody eluted.


No eluted target protein detected

Target protein not expressed in sample used/Low level of target protein expression 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.

Insufficient antibody for capture of the target protein
Check that the recommended amount of antibody is being used.

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 immunoadsorbent beads
Ensure you are using the correct beads for the antibody isotype used.


Immunoprecipitation - References


Using Antibodies: E.D. Harlow & D. Lane; Cold Spring Harbor (New York) (1999)

Current Protocols in Immunology: J.S. Bonifacino, et al.; New York (John Wiley) Sections 8.3.1-8.3.28 (2001)

Chromatin Immunoprecipitation Assays: Methods and Protocols: P. Collas;  Methods Mol. Med. 567, 1-258 (2009)


Immunoprecipitation - Online Resources


http://en.wikipedia.org/wiki/Immunoprecipitation

http://www.animal.ufl.edu/hansen/protocols/imp98.prt.htm

http://www.molecularstation.com/protein/immunoprecipitation/

http://pingu.salk.edu/~sefton/Hyper_protocols/immunoprecip.html

Tips and Troubleshooting