Biology Reference
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VI. Single-step Immunoprecipitation
Immunoprecipitation using specific antibodies is a powerful method for analyz-
ing protein-protein interactions and identifying protein complexes. Single-step
purification using polyclonal antibodies followed by mass spectrometry on the entire
eluate commonly indentifies several hundred to a thousand proteins making it
challenging to distinguish between signal and noise. Background can arise from
general non-specific binding to beads, to constant regions of antibody chains, and to
partially denatured antibodies. Background can also be specific to individual affin-
ity-purified antibodies - consequently a random IgG control cannot be used to
discriminate between true signal and noise. We recommend using as a control a
polyclonal rabbit antibody raised against GST (glutathione S-transferase) that is
affinity-purified using the same procedures used for the antibody to the target
protein. In addition, whenever possible, we recommend parallel immunoprecipita-
tions with either two antibodies to the same protein (preferable with non-overlapping
epitopes) or one antibody and one tagged fusion protein. Purification from a mutant
strain is an ideal negative control but is not feasible if the mutation is lethal. If a
tagged protein is immunoprecipitated, an untagged strain can be used as a negative
control.
The following two examples illustrate how redundant purification strategies and
suitable negative controls helped pinpoint bona fide complex members. Polyclonal
rabbit antibodies to two essential chromosome segregation proteins, KNL-1 and
KNL-3, identified in an RNAi screen, were used to purify complexes containing
these proteins ( Cheeseman et al., 2004; Desai et al., 2003 ). Each protein was present
in the other immunoprecipitation, allowing cross-referencing of the two immuno-
precipitations to identify 11 proteins in common ( Fig. 8 ) (in total
the two
Fig. 8 Identification of 10-protein kinetochore complex. Immunoprecipitation of KNL-1 and KNL-3
isolated more than 130 interacting proteins with 11 proteins common in both immunoprecipitations. MIS-
12 and KBP-1, two common interactors, were LAP tagged and purified isolating 11 common interactors
of which 10 were found also in KNL-1 and KNL-3 immunoprecipitations. (For color version of this figure,
the reader is referred to the web version of this topic.)
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