Hi George, if you don't want your eluted IPs to be have any Abs, you will have to attach your Abs directly to an activated resin (CNBr Sepharose, Affigel, etc.) .

If you use protein A/G resins to attach your Abs, most likely the antibodies will come off the resins in your elution steps. 

Thermoscientific Pierce has a Crosslinking CoIP kit. Ideally the crosslinked antibodies shouldn't come off during elution.

Thanks a lot for all the input. Actually I have already used the crosslinking coip kit from Pierce, but still I see heavy contamination with IgG chains. I followed the protocol precisely and I just played with amount of antibody. I will revisit the procedure and see what was wrong

Hi George, what is the elution buffer you are using?  

Hi Steingrimur. After extensive washing with buffer from kit I normally boil beads with Laemmli sample buffer (for routine work; custom made) but I also dissociate immune complexes with acidic 0.2 M glycine (also custom made) but in every case I see huge heavy and light chain IgG bands. I know that I must be doing something wrong, but I have no idea what. I can return to Protein G beads but proteins we need to pull down are in similar MW range (25-50 kDa) with antibody chains

Hi George if you are using the coip kit from Pierce, you could try loading up the resin with your Ab, do the crosslinking with the kit and afterwards treat it with the glycine buffer to remove any uncrosslinked Abs. before you apply your sample to the resin and do the IP.

Just to note, the DSS crosslinker in this kit is very labile. You have to use it within minutes of dissolving it. If you try to store  it in a solvent that has any H2O in it, it will degrade really quickly and become useless.   

Hi Steingrimur and apologies for late response. These are very helpful hints. Thanks a lot. I am going to try them. Do you think it might be possible to substitute crosslinker with some alternative?

Hi George, no prob.

I've attached some publications on crosslinking Abs to protein A resin:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2643637/

http://www.acsu.buffalo.edu/~lread/pdf/2001molbiochemparasitol.pdf

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773615/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1133783/

The main thing from these publications is that many of them use dimethylsuberimidate as a crosslinking agent, and that they extensively wash the antibody resin before using it.

Hope this helps you.

Hi,

Dynabeads Antibody Coupling Kit (143.11)

Dynabeads and co-IP - 143.21D

These kits were used in the following references:

Nucleic Acids Research, 2014, Vol. 42, No. 14 9021

Methods and Protocols, Methods in Molecular Biology, vol. 1102, 2014

PLOS ONE | March 2014 | Volume 9 | Issue 3 |

PLOS ONE | January 2014 | Volume 9 | Issue 1 |

kind regards

ketil

Hi, 

For covalent coupling the Dynabeads M-280 Tosylactivated (142.03) or Dynabeads M-270 Epoxy (143.01) are good alternatives. There is also a ready made kit (Dynabeads® Antibody Coupling Kit, 14311D) which includes both surface activated Dynabeads and buffers for covalent coupling. An additional alternative is available for co-IP (Dynabeads® Co-Immunoprecipitation Kit Cat. No. 143-21D) which contains both beads and buffers for covalent coupling and buffers optimized for co-IP (see ref Alber et al., (2007) Determining the architectures of macromolecular assemblies Nature 450, 683-694).

For covalent coupling there are several alternatives such as:

Dynabeads M-280 Tosylactivated (142.03) (stand alone Dynabeads - no buffers included)

Dynabeads M-270 Epoxy (143.01) (stand alone Dynabeads - no buffers included)

Dynabeads® Antibody Coupling Kit, 14311D) (Dynabeads M-270 Epoxy + buffers for covalent coupling)

co-IP (Dynabeads® Co-Immunoprecipitation Kit Cat. No. 143-21D) (Dynabeads M-270 Epoxy + buffers for covalent coupling + buffers optimized for co-IP (see ref Alber et al., (2007) Determining the architectures of macromolecular assemblies Nature 450, 683-694).

The Co-IP Kit consists of 2 parts:

•         Covalent Ab coupling to Dynabeads M270 epoxy

>        Dynabeads benefits

>        Freedom to use any Ab

>        No release of Ab in the eluate

•         Co-IP buffer system

>        Easy optimization

>        Qualitative reproducibility

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

• Salts

• Detergents

• Dithiothreitol (DTT)

• Protease Inhibitors

Buffer modifiers

• Sodium Chloride (e.g., 1 M NaCl)

• Dithiothreitol (e.g., 1 M DTT)

• Magnesium Chloride (e.g., 1 M MgCl2)

• Potassium Acetate (e.g., 1 M KOAc)

• Tween-20

• Triton X-100

• Protease inhibitors without EDTA

Kind regards

Ketil