Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 291

Before starting
Pipet 15 m L water into two screw-cap tubes, mark the liquid level, and remove the
water.
Put distilled water in sonicating water bath (Bransonic Ultrasonic Cleaner 3510)
and turn heating to 80 C or to maximum. If the sonicating water bath does not have
heating capability, boiling water should be added prior to use.
1. Transfer 30 RNAi and 30 control worms into 0.5 mL M9 in marked tubes. We
typically use injection as a method for RNAi as it has the best penetrance for early
embryonic depletions, but soaking or feeding can also be used.
2. Add 1 mL M9 and pellet at 200g in a microcentrifuge. Carefully remove super-
natant leaving the worms undisturbed.
3. Repeat step 2 two times.
4. After last wash, remove all liquid down to 15 m L mark and add 15 m L2 Sample
Buffer.
5. Place in sonicating water bath at 80 C. Sonicate on maximum setting for
15-20 min.
6. Microcentrifuge at 200g and check that worms have dissolved (you should not
see a pellet). If a significant pellet remains boil the samples at 95 C in a heat
block with intermittent vortexing.
7. Lightly centrifuge, mix by flicking, and load 10 m L/lane for immunoblots (aim
for one worm per microliter of final sample).
C. Tandem Affinity Purification Tags
Tandem Affinity Purification allows the isolation of protein complexes in high
purity. A composite tag is fused to the protein of interest containing two different
purification tags separated by a protease cleavage site ( Fig. 3 ). The original TAP tag
used a domain of protein A that binds to IgG and a calmodulin-binding peptide
(CBP) separated by a highly specific tobacco etch virus (TEV) protease site
( Rigaut et al., 1999 ). In C. elegans, a version of this approach that we have used
with significant success is the localization and affinity purification (LAP) tag
( Cheeseman and Desai, 2005 ). The LAP tag is a modification of the TAP tag that
can be used to both affinity purify the fusion protein and study its in vivo localization
dynamics. The LAP tag contains GFP (or mCherry) and S peptide (that binds
S protein with high affinity) separated by a TEV cleavage site ( Fig. 3A ). The
LAP-tag fusion protein is first purified using anti-GFP-coupled beads, released
from the beads by TEV protease cleavage and further purified in a second step over
S protein agarose. The LAP tag has been successfully used in several studies to
isolate new protein complexes ( Audhya et al., 2005; Cheeseman et al., 2004;
Dammermann et al., 2009; Gassmann et al., 2008 ). When using this tag for analysis
of protein complexes containing RNA, it should be kept in mind that the binding of S
peptide to S protein reconstitutes an active ribonuclease.
Several LAP-tag containing vectors are available: pIC26 allows fusing the target
protein at its N-terminus to the LAP tag using a SpeI restriction site ( Fig. 3B-D )