Immunodepletion is a method for removing a target molecule from a mixture. One common application is to split a complicated mixture (such as serum, cell lysate, homogenized tissue or conditioned media) into two batches, then subtract a targeted molecule from one batch by immunodepletion. The control and depleted batches can be then tested side-by-side in a biological assay and the effect of the depleted compound is discerned by comparison. Depletion typically begins by adding an antibody targeting the molecule of interest and then following with a solid phase material to bind the antibody. Examples include beads (agarose or magnetic) conjugated to protein A, protein G, avidin or anti-IgG. After mixing, the samples are centrifuged and the supernatant is saved as the depleted (or control) product.
Important considerations:
The efficiency of immunodepletion will vary dramatically from antibody to antibody. Some antibodies may not work at all.
Use purified antibody. Antibodies still in ascites fluid or in serum will contain numerous impurities that will confound results.
Generally speaking, the more antibody, the more efficient depletion. This is NOT like Western blotting. Expect to use antibody at concentrations of a few micrograms per ml to up to 100 ug/ml.
Use excess bead conjugate. Manufacturers assume ideal conditions when describing antibody binding capacities of their beads. A three to four fold excess is recommended.
When immunodepleting serum or serum-containing solutions, the use of a biotinylated primary antibody and the use of avidin or streptavidin agarose will avoid precipitating endogenous antibodies.
Of course, effective depletion cannot be assumed. Western blotting with depleted and undepleted solutions side-by-side proves efficacy.
Do not expect 100% depletion. A reduction of 90% or more is quite good.
Protein Mods offers a number of proteins conjugated to agarose for your immunodepletion needs.
Negative controls available in our catalog:
