DETECTION AND ANALYSIS OF PROTEINS - A Guide to Methods in the Biomedical Sciences - page 22

labeled by metabolic labeling so that the size, and other properties, of
the other interacting components can be determined following immuno-
precipitation using autoradiography. As in all studies of protein-protein
interactions, artifacts can occur and additional studies to confirm that
co-immunoprecipitated proteins indeed interact in vivo are always war-
ranted.
Far western blot
The far western blot is a modification of the western blot designed to
study protein-protein interactions. As in a western blot, proteins (usually
from cell lysates) are first resolved on the gel of choice (reducing SDS-
PAGE or native PAGE) and then transferred to a membrane. The prey
proteins on the membrane are probed with a known protein of direct
visualization, or identified using a secondary antibody. If the bait protein
is bound to one or more proteins on the membrane, it will then be identi-
fiable. Far western analysis can also be done “in-gel” by first renaturing
the resolved prey proteins in the gel and then using the bait to probe
the gel.
Pull-down assays
The pull-down technique has become a valuable tool to identify inter-
acting proteins. It can be used to confirm previously suspected interac-
tions suggested from results of co-immunoprecipitation studies, results
from non-denaturing gels or density gradient analysis, etc. or used to as
a screening assay to search for previously unknown interacting partners
for a protein of interest. Pull-down assays require that one of the inter-
acting partners, the bait, be available in purified form. This is usually
accomplished using recombinant DNA techniques. In addition, the bait
must be tagged in some way. It can be tagged with biotin (hence, biotin
pull-down assays) or as a fusion protein with a sequence tag produced
using prokaryotic or eukaryotic expression system (see Chapter 3). The
bait is then used to interact with prey proteins obtained both the bait
and its interacting partners. The interacting partners can then be re-
solved on SDS-PAGE and identified by staining the gel and/or western
blotting, etc.
Cross-linking agents
While many proteins can be isolated by one of the methods de-
scribed above, many functional protein interactions are only transient,
making their identification difficult. This is where cross-linking reagents