Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 290

+ 2% BSA + 0.1% Triton X-100 + 0.1% sodium azide) with 100 m LofE. coli
protein agarose for 1-2 h at room temperature. The supernatant is then mixed with
50 m g/mL final concentration of an unrelated fusion protein purified from bacteria
(harboring the same tag as the antigen) and then used for immunoblotting. This
procedure eliminates anti-bacterial protein antibody cross-reactivity even in highly
sensitive chemiluminescent detection.
B. Assessing Antibody Specificity by Immunoblotting after RNAi
The following protocol describes how to prepare worm extract for immunoblot
analysis to test antibody specificity. It is important to wash the worms thoroughly to
remove bacteria. Worms can be washed for up to 2 h in M9 containing 0.1% Triton
X-100 if bacterial contamination remains a problem. As noted above, a good way to
prevent bacterial epitopes in the sample is to use embryonic extract. However, a
tradeoff with using embryo extract is that RNAi has to be performed by feeding,
which might be less penetrant than injection. Therefore, we routinely perform RNAi
blots using worms and, if necessary, treat the primary antibody to remove/block
antibodies to bacterial epitopes.
To determine RNAi efficiency, a serial dilution of extract prepared fromwild-type
worms should be analyzed on the same blot as the RNAi extract sample ( Fig. 2 ). As a
loading control, a primary antibody of a different species should be used - we
typically use anti a -tubulin antibody that was generated in mouse (DM1A Sigma-
Aldrich, Cat. #T9026).
RNAi-mediated depletion of gene products has shown to be effective for a large
number of genes, including essential genes ( Green et al., 2011; Kamath et al., 2003;
Sonnichsen et al., 2005 ). If reduction of the band detected by western blotting is not
observed after RNAi this may be due to low RNAi efficacy or due to the antibody
recognizing a non-specific band of similar molecular weight as the gene product of
interest. In this case, alternate RNAi conditions (feeding, injection, soaking, tem-
perature, and time) or, ideally, null mutants should be analyzed by western blotting to
assess antibody specificity.
Fig. 2 Immunoblot of wild-type and knl-1(RNAi) adult extract probed using anti-KNL-1 and anti
a -tubulin antibodies. A serial dilution of wild-type extract was loaded to determine the efficiency of
KNL-1 depletion.