Immunoprecipitation Troubleshooting Guide

A well-planned experiment, with appropriate controls, treatments, and conditions, is often the first step toward obtaining improved results. To learn more about planning your Immunoprecipitation (IP) experiments, check out our Immunoprecipitating Experimental Guidelines.

Problem	Possible Causes	Discussion	Recommendation
Low/No signal	Protein-Protein Interactions Disrupted by Stringent Lysis Conditions	If no signal is seen in your co-IP experiment, the lysis buffer used may be disrupting protein-protein interactions. While both Cell Lysis Buffer #9803 and RIPA Buffer #9806 are suitable for generating whole cell extracts for western blotting, we only recommend the #9803 Cell Lysis Buffer for IP or co-IP experiments. RIPA Buffer is regarded as a stronger denaturing buffer as it contains the ionic detergent sodium deoxycholate. This detergent helps to disrupt nuclear membranes and further solubilize cellular and membrane components, while preventing protein degradation and not interfering with immunoreactivity. RIPA buffer has been known to denature kinases and prevent protein-protein interactions and is therefore not suitable for co-IP.	There are many factors involved that can affect the outcome of the assay, such as the strength of interaction of the associated proteins, and oftentimes the researcher may need to adjust their experimental conditions for optimal results. Cell Lysis Buffer #9803, along with the protocol we recommend for performing IPs, would serve as a suitable starting point for performing Co-IPs. Please keep in mind that sonication is crucial when using either of the aforementioned lysis buffers. It will ensure ample nuclear rupture, DNA shearing, and will lead to the greatest potential protein recovery. Sonication is particularly important to extract nuclear and membrane proteins and will not disrupt most protein complexes. Include an input lysate control to ensure that the target protein is expressed at high enough levels for detection in your samples and that the antibody to your target of interest is working properly. Probe the blot with an antibody to your IP protein to ensure that the IP experiment worked and that you successfully pulled-down your primary protein.
	Low Protein Expression in Tissue or Cell Line	If no signal is seen in your IP experiment, the IP or interacting proteins of interest may be expressed at levels below the level of detection for western blot.	Use expression profiling tools such as BioGPS or The Human Protein Atlas as well as scientific literature to check whether or not your cells or animal tissues are expected to sufficiently express the target protein of interest. We always recommend including a known positive control to confirm experimental results. A list of recommended controls for many of our antibodies can be found on our Control Treatments by Target table. Include an input lysate control to ensure that the target protein is expressed at high enough levels for detection in your samples and that the antibody to your target of interest is working properly.
	Low Level of Phosphorylated or Modified Protein	Many post-translationally modified proteins are expressed at low basal levels in cell lines or tissues. Some targets may require additional treatment with chemical modulators or growth under specific conditions to be expressed at levels high enough for western blot detection.	We recommend using PhosphoSitePlus to look up low-throughput papers referencing your particular modification site or use our Control Treatments by Target table to find an example of a treatment and cell line or tissue that works well as a positive control. Inclusion of phosphatase inhibitors in the cell extract is essential to maintain protein phosphorylation. Sodium pyrophosphate (2.5mM final concentration) and beta-glycerophosphate (1.0mM final concentration) should be included as serine/threonine phosphatase inhibitors in the lysis buffer. Sodium orthovanadate (2.5 mM final concentration) should be included to inhibit tyrosine phosphatases. Phosphatase Inhibitor Cocktail #5870 or Protease/Phosphatase Inhibitor Cocktail #5872 may also be used. Include an input lysate control to ensure that the target protein is expressed at high enough levels for detection in your samples and that the antibody to your target of interest is working properly.
	Epitope Masking	Epitope masking is when the antibody's binding site on the target IP protein is obscured either by the target's conformation under native conditions or by other interacting proteins, leading to negative IP results.	If epitope masking is suspected, it is best to try an antibody that recognizes an epitope in a different region of the target protein. Information about the epitope region for CST antibodies can be found under the Source / Purification section on the antibody product page.
	Low Binding of IgG to Beads	While both Protein A and Protein G beads can be used successfully with rabbit and mouse antibodies, CST recommends using Protein A beads when working with rabbit antibodies and Protein G beads when working with mouse antibodies. This is due to the fact that Protein A beads have a higher affinity for rabbit IgG and Protein G beads have a higher affinity for mouse IgG.	Optimize the bead choice according to the host species of the antibody being used for the IP. Combination Protein A/G beads may also be helpful in increasing IgG binding.
Multiple Bands or Non-specific Binding	Non-Specific Binding of Off-Target Protein(s) to Beads or IgG Control	If non-specific background signal is observed in your IP experiments, it may be caused by off-target proteins binding to the Protein A or G on the beads or by IgG directly.	A bead-only control acts as an additional negative control to account for any non-specific protein-bead interactions within an IP experiment. If background is observed in the bead-only control experiment, preclearing the lysate may be necessary. Preclearing is performed by incubating the lysate with beads alone for 30-60 minutes at 4°C prior performing the IP experiment. An isotype control may also be included to show if the background is caused by protein binding non-specifically to the IgG of the IP antibody.
	Isoform Reactivity or Post-Translational Modifications	Some cell line and tissue models can contain more than one protein isoform or splice variant, which can migrate at different molecular weights. Post-translational modifications (PTMs) may cause a subset of the target protein to run at a different rate than the unmodified protein. Glycosylation, SUMOylation, ubiquitylation, and phosphorylation are examples of modifications that can cause multiple bands to appear on a western blot, depending on the samples and treatments used.	Include an input lysate control to determine if the additional bands are caused by binding to the antibody. Refer to the Specificity / Sensitivity section on the antibody's webpage to determine if it is predicted or confirmed to detect more than one isoform. You may also reference your protein of interest on UniProt to see if multiple isoforms sequences are listed. If you are looking for more information on PTMs associated with your target protein, please see PhosphoSitePlus. If the background is not observed in the input control, then the source may be due to non-specific binding of proteins to the beads or IgG. In this case, a bead-only or isotype control will be necessary to determine the cause of the additional bands (see above section).
Masking of Target Signal by IgG	Target Signal Obscured by Heavy or Light Chain IgG	When performing an IP followed by western blotting, the denatured IgG light and heavy chains of the primary antibody used for the IP run at approximately 25 and 50 kD, respectively, on the western blot and can often obscure target proteins bands of a similar molecular weight. The use of antibodies from the same host animal for both the IP and western blot is the cause of this result. The secondary antibody used in the western blot not only detects the native IgG used for the primary antibody incubation, but also detects the denatured heavy and light chains of the antibody used for the IP.	There are several methods to avoid this outcome: Use antibodies from different species for the IP and the western blot. For example, using a rabbit antibody for the IP and a mouse antibody for the western blot or vice versa. Please note that the secondary antibodies need to be species-specific. Our testing has been performed with Anti-rabbit IgG, HRP-linked Antibody #7074 and Anti-mouse IgG, HRP-linked Antibody #7076, which are highly specific for rabbit and mouse, respectively. We have not observed species cross-reactivity with these secondaries. Use a biotinylated primary antibody from the same species for the western blot. The biotinylated antibody can then be detected with Streptavidin-HRP #3999, which will not cross-react with denatured IgG. If your target does not migrate near 25 kDa, a light chain specific secondary antibody can be used. CST offers a Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) #93702 and a Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) #58802. Replace the secondary antibody in the western blot with our Protein A (HRP Conjugate) #12291, which preferentially binds native IgG. However, it is important to note that #12291 may cross-react with denatured IgG at high concentrations. Use a conformation-specific secondary antibody for the western blot. CST offers a Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb #5127, which preferentially binds IgG in its native conformation. However, it is important to note that #5127 may also cross react with denatured IgG at high concentrations.