Biology Reference
In-Depth Information
affinity complexes (
Rigaut et al., 1999
). Two sequential affinity purification steps
significantly reduce background and lead to clean isolation of protein complexes.
Two potential drawbacks of this approach are that the tag has the potential to alter
protein function and that the stringency of the two-step purification procedure may
cause loss of low affinity interacting proteins. As either strategy has drawbacks,
whenever possible we conduct both in parallel. Such a dual approach was critical
in defining the protein network that constitutes the core microtubule-binding site
of the chromosome during cell division (
Cheeseman et al., 2004; Desai et al.,
2003
).
Below we discuss first the tools necessary for biochemical analysis of a protein of
interest followed by detailed methods for large-scale worm culture, extract prepa-
ration, and protein complex isolation (
Fig. 1
). We additionally profile methods to
assess specificity of antibodies and optimize starting material
for complex
isolations.
II. Generating Tools for Biochemistry
A. Generating a Polyclonal Antibody
We highly recommend that an affinity-purified antibody be generated to every
protein of interest. An antibody that recognizes its target with high specificity is an
invaluable reagent, and in most cases will be suitable for immunoprecipitation,
localization studies, and immunoblotting. Ideally, two independent antibodies
should be generated against non-overlapping epitopes. We have had good success
with soluble fusion protein fragments of 100-200 amino acids expressed in bacteria,
purified, and sent to a commercial vendor for antisera production (almost always in
rabbits). The antibody is affinity purified from the antisera using columns with
immobilized antigen that lack the fusion tag used for the initial antigen purification.
Alternatively, antibodies against the tag can be first depleted from the antisera prior
to affinity purification. Peptide antibodies can also be produced but, in our experi-
ence, have significantly lower rates of success than fusion protein antibodies. A
newer option is DNA-based immunization, which does not require antigen purifi-
cation for immunization but still requires purified protein for affinity purification of
the antisera (
Chowdhury, 2003
).
The specificity of the affinity-purified antibody must be validated by immunoblot
and immunofluorescence using wild-type and mutant backgrounds. If a mutant is not
available, RNAi targeting the protein of interest should be performed. The most
common cross-reactivity we have observed in fusion protein injection-generated
antibodies is to E. coli proteins that are present in both the injected antigen and in the
antigen preparations used for affinity purification. Because worms eat E. coli,
bacterial protein epitopes are difficult to eliminate. If the protein of interest is
present in embryos, the use of embryo isolated by bleaching avoids this problem
as these do not have bacterial epitopes. Contaminating antibodies to bacterial pro-
teins can be depleted using immobilized E. coli
lysate (Thermoscientific Cat.
Search WWH ::
Custom Search