Could be due to one of the many possiblities :

1. The antibody may not be specific. (Are you able to identify your protein clearly by Western Blot or ELISA using the same antibodies ? This is just to know how specific your antibody is to your protein. If the antibody fails in this step then you wont be able to pull down the target protein; let alone the interacting partner.)

2. Binding Buffer conditions were not compatible. (Sometimes the salt or the detergent concentrations in the kit's binding buffer may be too much to allow for your antibody-target interaction. In this case you can prepare the same buffer yourself and reduce the salt (to as low as 100 mM NaCl and not lower) and/or detergent concentration (to as low as 0.5% NP40). You need to be careful about reducing these factors too much as the chances of getting non specific interactions will increase. So reduce stepwise and aim to find the sweet spot.)

3. Target protein is degraded or proteolysed (Make sure protease inhibitors are fresh and added to your lysis buffer. Always maintain 4C)

4. The proteins were lost to washing post-binding. (Atleast till you get a positive interaction with the target, wash twice with the binding buffer only. Once you start seeing positive results you can become more stringent with the wash conditions)

You dont really need to incubate the beads with your lysate overnight, especially to identify interacting partners. Proteins in the lysate with low zeta potential will begin to aggregate the longer you leave it and a lot of non specific proteins will then pull down with your beads. Binding at 10 to 20 mins at RT or 30 mins to 1 hour at 4C should be more than enough.

Aim to elute your protein using low pH Glycine or high pH triethanolamine for samples you validate by SDS-PAGE and silver staining/WB. Boiling is fine, but you run the risk of getting heavy and light chain antibody bands in your gel. If any of your potential Co-IP proteins run at the same size as the antibody bands, they will get masked. But if you are planning to send the sample for LC/MS, then boiling wouldnt make any difference.

Could be due to one of the many possiblities :

1. The antibody may not be specific. (Are you able to identify your protein clearly by Western Blot or ELISA using the same antibodies ? This is just to know how specific your antibody is to your protein. If the antibody fails in this step then you wont be able to pull down the target protein; let alone the interacting partner.)

2. Binding Buffer conditions were not compatible. (Sometimes the salt or the detergent concentrations in the kit's binding buffer may be too much to allow for your antibody-target interaction. In this case you can prepare the same buffer yourself and reduce the salt (to as low as 100 mM NaCl and not lower) and/or detergent concentration (to as low as 0.5% NP40). You need to be careful about reducing these factors too much as the chances of getting non specific interactions will increase. So reduce stepwise and aim to find the sweet spot.)

3. Target protein is degraded or proteolysed (Make sure protease inhibitors are fresh and added to your lysis buffer. Always maintain 4C)

4. The proteins were lost to washing post-binding. (Atleast till you get a positive interaction with the target, wash twice with the binding buffer only. Once you start seeing positive results you can become more stringent with the wash conditions)

You dont really need to incubate the beads with your lysate overnight, especially to identify interacting partners. Proteins in the lysate with low zeta potential will begin to aggregate the longer you leave it and a lot of non specific proteins will then pull down with your beads. Binding at 10 to 20 mins at RT or 30 mins to 1 hour at 4C should be more than enough.

Aim to elute your protein using low pH Glycine or high pH triethanolamine for samples you validate by SDS-PAGE and silver staining/WB. Boiling is fine, but you run the risk of getting heavy and light chain antibody bands in your gel. If any of your potential Co-IP proteins run at the same size as the antibody bands, they will get masked. But if you are planning to send the sample for LC/MS, then boiling wouldnt make any difference.

Hi there,

As mentioned above, there might be multiple issues. I would advise to analyze every step of the process by WB. The main issue being the presence of the protein of interest from the start and how stable it could be through the whole process.

Following the answer to this interesting query.

I had the same issues with my protein of interest. I was trying to map out an interaction between two proteins. But, there was no target protein eluted on the WB. I checked following things,

a. Labile interactions are so sensitive to be captured. Hence, I performed live-cell crosslinking with DSP to make the interactions strong enough and kept all the samples at 4 C throughout the experiment.

b. I incubated the Ab with beads for 15 mns at RT and performed IP for a short-time 20 mns at RT for antigen-antibody binding rather than at 4 C for ON (Note: I couldn't see my protein of interest at ON incubation).

c. I even saved the supernatants after the antigen-antibody binding (after 20 mns) and loaded some amounts of it.

c. I looked for bait protein on the WB after IP to be sure that IP to the bait is successful and also to know that if there is any problem with the beads and antibody binding to the bait protein.

d. I checked a positive control (the protein is known to be interacting with the bait protein) on the WB to check that the IP to bait is working fine.

e. Glycine elution was performed and the samples were boiled for 5 mns in the presence of sample buffer and loaded on to the gel.

f. I even performed a time course of stimulating the cells. The interaction differs based on the stimulation and time points of stimulation if you work with the cells lines especially.

Finally, I could see my protein of interest coming up in the elution. The protocol might vary for you a little bit. But I would suggest you to go through all of the steps once again to be sure that there are no mistakes from your hands.

Are you getting your bait protein in the input or lysate before IP ?? If yes, then first rule out if there is problem in binding with dynabeads. The protein A/G dynabeads work better than Protein G only. Also, as others suggested, the overnight incubation of bead with protein extract/lysate may be just too much for optimal binding and recovery. Try 20 min to 1 hour incubation at RT.

Best of luck

hi,

due to very rapid binding kinetics, the binding of the prim Ab to the beads is done within as little as 10 min. in most cases, extending the incubation time will not add more Abs to the bead surface. by extending the time to 1h or O/N did not result in more Abs on the beads.

Factors that we know are important are

- mixing conditions during washing (to rough mixing result in loss of binding)

- number of washing steps (too many - loss of binding)

- elution volume (increased yield of target with increase volume)

As mentioned by Tyagi above, protein protein interactions can often be very weak and transient and can therefore be challenging to capture. the buffer composition needs to be optimized in order to have the optimal stringency. for targets which are rare, an indirect approach can be tested as well - extending the incubation time. in terms of longer incubation times it is always a balance between capturing the target and adding more unspecific binding to the system. most of the specific binding will take place very fast.

An alternative to the Dynabeads Protein G is to covalently conjugate the Abs to the e.g. Dynabeads M-270 Epoxy. The Abs will not fall off during mild elution - only the target complex.

Components that can be varied to increase or decrease stringency of the Co-IP include;

• Salts, Detergents, Dithiothreitol (DTT), Protease Inhibitors

kind regards

ketil