Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support—Troubleshooting

The light chains or heavy chains of IgG could migrate at the same molecular weight as your protein of interest under denaturing/reducing conditions, and give a signal on the western blot. To eliminate this problem, we recommend covalently attaching the IgG to a resin so that after washing and eluting, only the antigen is recovered from the resin.

For purified IgG, you could use our AminoLink™ Plus coupling chemistry with the Pierce™ Direct IP Kit (Cat. No. 26148, agarose-based) or our NHS-ester coupling chemistry with the Pierce™ Direct Magnetic IP/Co-IP Kit (Cat. No. 88828, magnetic bead-based), and for non-purified IgG, you could use the Pierce™ Crosslink IP Kit (Cat. No. 26147, agarose- based) or Pierce™ Crosslink Magnetic IP/Co-IP Kit (Cat. No. 88805, magnetic bead-based)). Additionally, using Clean- Blot™ IP Detection Reagent (Cat. No. 21230) or Clean-Blot™ IP Detection Kit (Cat. No. 21232) to detect only the native antibody that is used as the western blot probe will prevent detection of the denatured light/heavy antibody chains. Please note, that when using Clean-Blot™ IP Detection Reagent, an immunoprecipitation with a no antigen negative control should be included on the western blot to confirm that the IgG is not leaching off the resin and getting recognized by the Clean-Blot™ IP Detection Reagent.

The tag is most likely being masked during the folding of the protein. Upon denaturation by heating in reducing SDS- PAGE sample buffer, the tag is no longer covered and can be detected by western blotting. In order to alleviate the issue, try binding the tagged protein to the agarose resin under denaturing conditions so that the tag is accessible for the antibody to bind it and purify the fusion protein.

Additionally, check the binding conditions to ensure that the recombinant target protein will bind to the antibody you are using.

Finally, the location of the tag may be critical. Depending on how the protein folds, moving the tag from one end of the protein to the other may improve accessibility, and therefore, binding to the column. Another feature to consider is that if the protein is truncated during expression, a tag at the C-terminus is less likely to be intact and function for affinity purification of the recombinant fusion protein. Moving the tag to the N-terminus may help with purification (and possibly downstream detection).

Make sure that non-coupled antibody is completely removed from the resin by washing the antibody-coupled resin with the elution buffer until no additional antibody elutes from the resin, as determined by protein assay or measuring the absorbance at 280 nm.

Also avoid using buffers that contain reducing agents during the IP or elution steps as this will reduce the antibody and fragments or antibody subunits that are not covalently linked will be eluted.

Make sure that the sample contains enough antigen by verifying its expression and/or lysis efficiency of the lysate by SDS-PAGE or western blotting.

Secondly, make sure the antibody solution does not contain amines or carrier proteins which could compete with the antibody for coupling to the resin. You can verify the antibody coupling by monitoring the flow-through and wash fractions (i.e., measure the absorbance at 280 nm or analyze by SDS-PAGE).

Finally, it is possible that a component in the IP Lysis/Wash Buffer interfered with antibody-antigen binding. To prevent this, perform the IP and washes using 1X Tris-buffered saline.

It’s possible that your antigen is sensitive to low pH and has become inactive during the elution process. Repeat the IP and use a high-salt, neutral pH elution buffer, such as the Gentle Elution Buffer (Cat. No. 21027). Refer to this Tech Tip for suggestions on formulations for other elution buffers.

The protein may be hydrophobic so you can dissolve it in coupling buffer containing up to 20% DMSO for most proteins. We highly recommend that you confirm the compatibility with DMSO prior to exposing large quantities of protein to this organic solvent.

The proteins on the bead surface may adhere to the plastic tube. To avoid this, after the quenching step, add 0.05% detergent e.g., Tween™-20 Detergent) to the washes

Note: Do not use detergent in the coupling step.

• Make sure all primary amine-containing components of the buffer (such as Tris or glycine) are completely removed by dialysis or desalting or the use of the Pierce™ Antibody Clean-Up Kit (Cat. No. 44600) before coupling to magnetic beads
• Make sure the antibody is mixed with the NHS-beads IMMEDIATELY after washing the beads. A delay may cause hydrolysis of the NHS off the resin which leads to low coupling.
• Finally make sure the pH of the non-amine containing coupling buffer used for coupling is between pH 7–9

The antibody will come off the beads during elution if the antibody is not crosslinked to the beads. The antibody is attached to the Protein A or Protein G through affinity binding similar to the binding that occurs between the protein (=antigen) and the antibody. When you break one of the affinity bindings, you typically will break the other affinity binding as well.

Crosslinking is never a 100% reaction. Some antibodies are not crosslinked and may come off with elution. You can perform a washing step with low pH directly after crosslinking to remove non-crosslinked antibodies. Remember to bring the pH back to normal before your immunoprecipitation step.

Alternatively, if reducing agents are included in the buffers, they could cleave the four chains of the antibody apart, releasing those chains not covalently attached to the beads.

If you are using reducing agents in the sample buffer before gel loading, try incubating the beads in a sample buffer without reducing agents. Reducing agents such as DTT or ß-mercaptoethanol will reduce disulfide bridges and result in release of antibody light and heavy chains. You may also elute the protein by lowering the pH, to leave the antibody bound to the beads.

Here are a few suggestions to try:

• Use more stringent washing buffer for washing
• Add a non-ionic detergent (Tween™-20 or Triton™ X-100) to the washing buffer, in concentrations between 0.01–0.1%
• If the beads are blocked before precipitation, add identical blocker to the washing buffer
• Increase the number of washing steps
• Prolong the washing steps
• Decrease incubation time (beads and sample)
• Try the indirect IP method
• Decrease the antibody concentration
• A pre-clearing step may be performed to remove molecules that nonspecifically bind to the Protein A/Protein G or the beads themselves

Streptococcal Protein G comprises two or three domains that bind to the constant Fc region of most mammalian immunoglobulin Gs (IgGs). Protein G binds to the interface between the second and third heavy chain constant domains of Fc, which is roughly the same binding site used by Protein A. Protein G comprises one α-helix packed onto a four- stranded β-sheet. Residues from Protein G that are involved in binding are situated within the C-terminal part of the α- helix, the N-terminal part of the third β-strand and the loop region connecting these two structural elements. The Protein G:Fc complex involves mainly charged and polar contacts, whereas Protein A and Fc are held together through nonspecific hydrophobic interactions and a few polar interactions.

Elution, both mild and denaturing, will destroy the binding between antibodies to Protein A or Protein G. The strong elution also breaks the bond between the heavy and light chains of the antibodies and therefore the antibodies will be seen on the gels. The IgG heavy chain is about 50 kDa, the IgG light chain is about 25 kDa, and the IgG whole complex is about 150 kDa. NOTE: Mild elution does not always break the affinity of IgG to Protein A or Protein G. And strong elutions only break the IgG chains apart if a reducing agent is present.

If you do not want to see the antibody on the gels, you may consider crosslinking the antibody and Protein G or Protein A, prior to the immunoprecipitation. Alternatively, you can also use different species of antibody for blotting to avoid seeing the primary antibodies use for IP. For example, if you use the monoclonal antibody for IP, you can blot the membrane using a polyclonal antibody that would not recognize the monoclonal antibody.

Finally, the Clean-Blot™ IP Reagent detects only the native IgG; it will not detect IgG that has been denatured in SDS- PAGE Sample Buffer.

The binding sites of your antibody have likely been altered by the crosslinking. When this occurs, your antibody will show reduced affinity or no affinity to its target antigen. Another consequence of crosslinking can also be increased affinity for unintended (nonspecific) targets. This is always a high risk with crosslinking, and it is a problem easily avoided by choosing another path to covalent antibody coupling. You can try using the Dynabeads™ Antibody Coupling Kit. This kit is a far superior solution for covalent antibody coupling to Dynabeads™ magnetic beads (compared to crosslinking with Dynabeads™ Protein A or G magnetic beads). The Dynabeads™ Antibody Coupling Kit is compatible with almost any antibody. It is designed specifically for covalent antibody coupling to Dynabeads™ magnetic beads. Unlike crosslinking, the Dynabeads™ Antibody Coupling Kit will not alter antibody specificity or affinity.

Protein G is coated onto hydrophilic beads. If your background is protein-mediated, then we normally suggest having a combination of blocking protein and non-ionic detergent both in the coupling and washing buffer to reduce nonspecific binding.

 Here are some suggestions:

• Wash the beads prior to use in 100 mM glycine at a pH of 11.3 followed by a wash with 200 mM glycine at a pH of 2.8 for a very short period. Immediately transfer the beads to PBS with 0.01% Tween™-20.
• Perform the elution in the low pH, glycine-based elution buffer recommended in the manual.
• Crosslinking of the antibody to the beads in order to covalently attach the antibodies to the beads, which will also crosslink protein G.

This kit is not recommended for use with antibodies that have been stabilized in glycerol. The antibody function may be severely affected. Consider dialyzing the IgG to remove the glycerol and then repeat the coupling process.

Here are a few suggestions you can try:

• Perform IP without crosslinking the antibody to the beads
• Reduce the amount of crosslinker used to covalently attach the antibody to the beads
• Try a different crosslinker
• To prevent co-elution of antibody, try one of our surface-activated Dynabeads™ magnetic beads; this allows you to conjugate the antibody to the beads directly, through covalent binding

Here are a few suggestions to try if you are getting low binding to the Dynabeads® protein A/G beads:

• Verify binding/specificity of your antibody to your antigen, e.g., by ELISA
• Check the binding of your antibodies to the beads; if the antibodies are not captured and bound to the beads, the immunoprecipitation experiment will not work
• If you have used the indirect method, try the direct method; conversely, if you have used the direct method, try the indirect method
• Check the amount of beads and sample volume; with reference to the capacity of different beads proposed in the package inserts, increase the amount of beads or the concentration of your antibody during coupling
• Increase the incubation time
• If using a commercially sourced antibody, confirm it is validated for IP
• Try another antibody

Here are some reasons why the immobilized “bait” partner may not pull down the interacting “prey” protein and some tips to improve the pulldown:

• It is possible that the prey protein is degraded: in this case, include protease inhibitors and or phosphatase inhibitors (see examples) in the lysis buffer. Also use fresh lysate or lysate frozen at –80 degrees C.
• If the interaction between bait and prey is weak or transient such that the stringent wash conditions disrupt the interaction, you can reduce the number of washes and ionic strength of wash buffer.
• If the prey protein is expressed at a low level, apply more protein (prey) sample, increase amount of bait protein and incubation time.
• Test to see if any cofactor is essential for the interaction being studied and include this in the incubation.
• It is also possible that binding or sample preparation conditions were insufficient to maintain or allow protein interactions. In this case, try alternative buffers for sample preparation, binding or washing procedures.
• Excessive labeling (e.g., biotinylation) of the bait could sterically hinder binding to prey protein: in this case reduce molar excess of the biotinylation reagent used for labeling or use a reagent that targets a different functional group.

After incubating with the lysate, you can increase the number of washes or increase NaCl concentration of the wash buffer. You can also add a small amount of non-ionic detergent (e.g., 0.2% of NP-40) to the wash buffer.

Alternatively, pre-clear the sample with control agarose to remove any proteins that could be nonspecifically binding and eluting with the proteins that are interacting.