Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a method used to determine the location of DNA binding sites on the genome for a particular protein of interest.

The principle underpinning this assay is that DNA-binding proteins (including transcription factors and histones) in living cells can be cross-linked to the DNA that they are binding.

The identity and quantity of the DNA fragments isolated can then be determined by polymerase chain reaction (PCR).

RIP and CLIP both purify a specific RNA-binding protein in order to identify bound RNAs, thereby studying ribonucleoproteins (RNPs).

In CLIP, cells are UV crosslinked prior to lysis, followed by additional purification steps beyond standard immunoprecipitation, including partial RNA fragmentation, high-salt washing, SDS-PAGE separation and membrane transfer, and identification of direct RNA binding sites by cDNA sequencing.

One of the major technical hurdles with immunoprecipitation is the great difficulty in generating an antibody that specifically targets a single known protein.

To get around this obstacle, many groups will engineer tags onto either the C- or N- terminal end of the protein of interest.

The advantage of an extremely high binding capacity must be carefully balanced with the quantity of antibody that the researcher is prepared to use to coat the agarose beads.

While some may argue that for these reasons it is prudent to match the quantity of agarose (in terms of binding capacity) to the quantity of antibody that one wishes to be bound for the immunoprecipitation, a simple way to reduce the issue of non-specific binding to agarose beads and increase specificity is to preclear the lysate, which for any immunoprecipitation is highly recommended.

In most cases, preclearing the lysate at the start of each immunoprecipitation experiment (see step 2 in the "protocol" section below)[6] is a way to remove potentially reactive components from the cell lysate prior to the immunoprecipitation to prevent the non-specific binding of these components to the IP beads or antibody.

[5] This approach attempts to use as close to the exact IP conditions and components as the actual immunoprecipitation to remove any non-specific cell constituent without capturing the target protein (unless, of course, the target protein non-specifically binds to some other IP component, which should be properly controlled for by analyzing the discarded beads used to preclear the lysate).

Monodisperse and polydisperse superparamagnetic beads are offered by many companies, including Invitrogen, Thermo Scientific, and Millipore.

The nature of magnetic bead technology does result in less sample handling[8] due to the reduced physical stress on samples of magnetic separation versus repeated centrifugation when using agarose, which may contribute greatly to increasing the yield of labile (fragile) protein complexes.

[8][9][10] Additional factors, though, such as the binding capacity, cost of the reagent, the requirement of extra equipment and the capability to automate IP processes should be considered in the selection of an immunoprecipitation support.

While these arguments are correct outside the context of their practical use, these lines of reasoning ignore two key aspects of the principle of immunoprecipitation that demonstrates that the decision to use agarose or magnetic beads is not simply determined by binding capacity.

So the decision to saturate any type of support depends on the amount of protein required, as described above in the Agarose section of this page.

The price of using either type of support is a key determining factor in using agarose or magnetic beads for immunoprecipitation applications.

This is because sepharose beads must be concentrated at the bottom of the tube by centrifugation and the supernatant removed after each incubation, wash, etc.

This imposes absolute physical limitations on the process, as pellets of agarose beads less than 25 to 50 μl are difficult if not impossible to visually identify at the bottom of the tube.

Conversely, spin columns may be employed instead of normal microfuge tubes to significantly reduce the amount of agarose beads required per reaction.

While clear benefits of using magnetic beads include the increased reaction speed, more gentle sample handling and the potential for automation, the choice of using agarose or magnetic beads based on the binding capacity of the support medium and the cost of the product may depend on the protein of interest and the IP method used.

Once this has occurred the immunoprecipitation portion of the protocol is actually complete, as the specific proteins of interest are bound to the antibodies that are themselves immobilized to the beads.

After washing, the precipitated protein(s) are eluted and analyzed by gel electrophoresis, mass spectrometry, western blotting, or any number of other methods for identifying constituents in the complex.