Immunoprecipitation is a technique to isolate a specific protein from a mixture using an antibody that binds to that protein. There are two main methods: traditional IP and crosslink IP. Traditional IP recovers both the antibody and antigen, which can make identifying the antigen difficult if they have similar sizes. Crosslink IP covalently attaches the antibody to beads, allowing isolation of just the antigen without antibody contamination to easily identify and analyze the target protein. However, in some rare cases the traditional method may work better, such as if the antibody is unstable or the antibody-antigen binding is too strong for elution.

1. Li Yinliang Alex

2.  Technique of precipitating a protein antigen out
of solution using an antibody that specifically
binds to the particular protein, so as to isolate
and concentrate a particular protein from a
sample containing different types of proteins.

3.  Traditional immunoprecipitation (IP) method
 Crosslink immunoprecipitation (IP) method

4.  Involves incubation of antibody with a protein
mixture containing the antigen, followed by
capture of the antibody - antigen complex to
Protein A or Protein G immobilized on beaded
agarose resin. After washing to remove
nonbound (presumably undesired) components
of the sample, antibody and antigen are
recovered by boiling the beaded affinity resin in
sample loading buffer for analysis by SDS-
PAGE.

5.  Uses the principle of crosslinking to covalently
attach the antibody to the Protein A/G Agarose
Resin – covalent immobilization of the antibody
to the agarose resin, enabling purification of
antigens without the usual co-elution of the
antibody heavy and light chain fragments.

6. Using the traditional immunoprecipitation (IP) method,
both the target antigen and the antibody are eluted in
the final fraction, resulting in the presence of prominent
heavy and light chain antibody bands in the final
electrophoresis gel. If the target protein has a similar
molecular weight to one of the contaminating antibody
fragments, its presence may be masked, making it
impossible to identify or further characterize the
immunoprecipitated product.
However using the crosslink immunoprecipitation (IP)
method, only antigen is eluted by the procedure,
enabling it to be identified easily (only one protein band
produced) and further analyzed without interference
from antibody fragments i.e. eliminating antibody
contamination as antibody is irreversibly attached to
agarose beads so that co-elution of heavy and light
chains with purified protein (antigen) is minimized. It
also increase yield of the method and antibodies used
may be reused again.

7.  Example.

8. However, there are certain atypical (uncommon)
circumstances that the traditional immunoprecipitation (IP)
method works better than the crosslink immunoprecipitation
(IP) method:
 Antibody used is very unstable and can be inactivated by the
covalent crosslinking step. Therefore as a result, there will
little/no antigen precipitated despite committing ample
(sufficient/excess) antibody to the procedure.
 The antibody:antigen affinity interaction are too strong that
elution buffer does not efficiently dissociate the target
protein (antigen) from the covalently immobilized antibody –
harsher elution conditions cannot be used, which may have
undesirable denaturing effects to the protein (antigen) of
interest.

9. Retrieved from website
 Wikipedia
 Piercenet