Hello Vusumuzi Ngonidzashe Hove

Yes, you are right in both cases.

If you incubate the protein lysate (that has the antigen of interest) with the antibody overnight followed by the addition of the antigen-antibody complex to the bead slurry the next day, you will get high purity of protein. However, the antibodies are also co-eluted with protein of interest which sometimes creates difficulties in western blot detection.

On the other hand, if you add the antibody to a bead slurry for a couple of hours followed by the addition of the antibody-bead complex to the protein lysate for incubation overnight, it will give you lesser yield than the above method, but you will avoid the problem of co-elution of antibodies.

I would prefer incubating the protein lysate (that has the antigen of interest) with the antibody overnight followed by the addition of the antigen-antibody complex to the bead slurry the next day.

Further, agarose beads, are extremely porous in nature which increases the risk that the antibodies will get trapped inside their sponge-like structure, making them inaccessible to the target proteins. Moreover, you need to ensure that the amount of antibody used is enough to coat the entire bead or you’ll increase the risk of getting elevated background signal due to the nonspecific binding of the lysate components to the unsaturated portions of the beads.

On the other hand, magnetic beads do not have a porous center that can help enhance their binding capacity. Their relatively small size (magnetic beads have a diameter of 1 to 4μm compared to agarose beads which have a diameter of 45 to 165µm) helps them achieve optimum antibody binding capacity. Magnetic beads don’t require huge amounts of antibody to produce accurate results. They are non-porous so you can be sure that the antibody will bind exclusively on the exposed outer surface of the beads.

So, it is better to use Protein A magnetic beads in comparison to protein A agarose beads for immunoprecipitation experiments.

Good Luck!

Malcolm Nobre thank you for the information it is very helpful much appreciated!

In addition to Malcolm Nobre magnetic beads have the big advantage of scalability in automated assay systems.

If you are setting up your assay on a small scale, and possibly have to optimize it anyway, begin with what you have or is easily available and switch to another system, if the results should not be satisfactory.

To avoid antibody related background and detection problems, you could use carboxy functionalized particles or sepharose instead of Protein A/G and covalently link your antibodies to them using EDC/NHS activation or directly off the shelf NHS activated sepharose.

Wolfgang Schechinger thank you for your input it is much appreciated.

Another option to avoid antibody-related background is the method we developed recently: https://www.mdpi.com/2409-9279/2/2/35

In this case, you get the high capacity and nice orientation of IgG by protein A or G and avoid the coelution of the antibodies by covalent cross-linking.

Michael G. Weller thank you for providing the paper for me to read I will definitely look into using the method you are referring to thank you.