Biology Reference
In-Depth Information
mixing of all proteins from the cells exposing the
protein of interest to cellular proteins that it would
never encounter under normal physiological
conditions. This issue can be partially offset by
subcellular fractionation of the cell prior to the
pull down experiment. If the protein of interest is
exclusively localized to the nucleus, a nuclear
extract can be used for the protein complex purifi-
or with a pool of nonspeci
c antibodies. The list
of proteins that are identi
ed in the control is
filtered from the experimental list. Any protein
that remains on the experimental list can then
be considered a potential speci
c interacting
protein. However, this method is not a very effi-
-
cient way to reduce the false discovery rate in an
af
-
cation minimizing the false interactions that occur
when a nuclear protein is exposed to the cytosolic
proteins. However, complete information
regarding the location of a protein and all of its
potential interacting proteins is rare; therefore,
restricting the pull down assay to only one cellular
compartment can bemisleading and result in both
false positive and negative discoveries. Another
important variable is the buffer used to lyse the
cell. The physical properties of proteins vary
signi
nity puri
cation experiment. There has been
signi
cant effort in addressing this dilemma
using novel experimental con
gurations and
constructing comprehensive databases cata-
loging all the proteins capable of binding to the
af
nity matrix.
The false negative discovery rate is also
a signi
cation
experiments. Many interactions are transient,
making them dif
cant problem in af
nity puri
cult to preserve throughout
the puri
cation procedure. It has been suggested
that by keeping the time for which the af
cantly; therefore, using only one experi-
mental condition can lead to signi
nity
reagents are incubated with the lysate to
a minimum might increase the likelihood of
preserving these interactions.
62
However, short
incubation times might not allow for the capture
of a suf
cant problems
as proteins will behave differently under different
buffer conditions. For example, a mild buffer
tailored to preserve interaction between hydro-
philic proteins will not work for highly
hydrophobic proteins because it will not be able
to solubilize them. Because the most common
practice during a high-throughput screen is to
use one experimental condition, it is expected
that it will not be suitable for all proteins. Further-
more,
cient quantity of the protein of interest,
causing diminished recovery of the protein
complex and missed protein
e
protein interac-
tions. Thus, it might be advantageous to perform
several af
cations at different time
points; however, this strategy is challenging
when performing a high-throughput pulldown
on multiple different proteins.
The selection of a physiologically relevant
cell line to perform the experiments that can
minimize the false negative discovery, as the
potential interacting proteins might not be
expressed in the heterologous cells typically
used for protein complex isolation. In addition,
the appropriate isoform or a required post-
translational modi
nity puri
the diverse physical properties of
the
proteins in the cell make it impossible to
nd
a single condition that will work for all proteins.
The most common way of liberating the
captured protein complex off the af
nity resin
is to boil the samples in a sodium dodecyl sulfate
(SDS)-based buffer. This action will release all the
pulled-down proteins,
including proteins that
nonspeci
cally interact with the
capturing
reagents. Unfortunately,
these proteins will
cation might not be
produced within the selected cell line leading
to a missed interaction. An alternative is to
use tissue samples that are known to express
the protein of interest. In this case, an antibody
wouldbeusedtotargettheendogenously
expressed protein. Unfortunately,
account for a signi
cant part of the nonspeci
c
background inherent to af
nity puri
cation. To
manage the background, a control af
-
cation experiment is performed in parallel. This
experiment is typically done on a lysate from
cells that do not express the protein of interest
nity puri
finding
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