TAP Purification from Worm Extracts
Initially described in Cheeseman et al. (2004). “A Conserved Protein Network Controls Assembly of
the Outer Kinetochore and Its Ability to Sustain Tension.” Genes & Development 18 (18): 2255-2268.
I. Generating LAP Tagged C. elegans strains
Clone either the complete genomic sequence or the corresponding open reading frame (i.e., cDNA)
into the SpeI site of pIC26 (see Fig. 1). Integrate pIC26 into C. elegans strain DP38 (unc-119 ed3) using
biolistic microparticle bombardment (see Praitis et al. (2001) Creation of low-copy integrated
transgenic lines in Caenorhabditis elegans. Genetics. 157:1217-26.). It is important to obtain a
homozygous strain as several generations of unselected growth are required to generate sufficient
material for a LAP purification.
pIC26 (pie-1::GFP-TEV-S; unc-119+)
Red – GFP
Black – Synthetic introns to improve expression of GFP fusion (see Fire et al. (1990). A modular set of
lacZ fusion vectors for studying gene expression in Caenorhabditis elegans. Gene. 93:189-98)
Green – Linker sequence
Dark Blue – TEV cleavage site (see Carrington and Dougherty (1988). A viral cleavage site cassette:
identification of amino acid sequences required for tobacco etch virus polyprotein processing.
Proc. Natl. Acad. Sci. U. S. A. 85:3391-5)
Light Blue – S peptide (see Kim and Raines (1993). Ribonuclease S-peptide as a carrier in fusion
proteins. Protein Sci. 2:348-56)
• Clone into the SpeI site (can use NheI or XbaI compatible ends if SpeI is present in gene of interest)
• Expression is controlled by pie-1 promoter and terminator which drive expression in the germline
• Vector is based on pAZ132 (see Praitis et al. (2001) Creation of low-copy integrated transgenic lines
in Caenorhabditis elegans. Genetics. 157:1217-26)
• Ampicillin Resistant
II. Preparing Adult Worms
Growing worms in liquid culture: Unsynchronized culture
Day 1: Axenize 4 plates of LAP Tagged adults. Spot 6 µl 2 M NaOH and 6 µl 100% bleach at edge of
bacteria on a small NGM agarose plate. Pick 10-15 adult worms into pool of liquid and allow to
dry. Grow until embryos hatch and become adults.
Day 5: Seed large plates with 15 cleaned LAP Tagged adults. You will need 3-5 plates per 500 ml of
liquid culture—put the plates at 20° C (4 days),[or at 24.5°C (3 days)]. Plan on starting 2 x 500 ml
of culture. Since it is highly likely that a couple of plates will become contaminated, set up slightly
more plates than you need.
Day 8: Start a 50 ml overnight of OP50-1 in LB + 50 µg/ml streptomycin.
Day 9: Start overnight cultures of OP50-1 in LB + 50 µg/ml streptomycin. You need 1 liter of culture
per 500 ml of C. elegans culture.
Day 10: Spin down bacteria in sterile 1 liter bottles. Resuspend the bacteria from each liter of culture
in 500 ml of S complete medium (see media recipes below) in a sterile 2.8 liter flask.
Look through the large plates under the microscope to make sure that NO contamination is present.
Rinse ~8 plates of LAP Tagged worms (just starved) each with 5 ml of sterile M9 into a 50 ml conical
tube. Chase with an additional 5 ml of M9 per 2 plates. Spin worms in a clinical centrifuge for 3
minutes at 600 g to pellet. Wash with 50 ml of cold M9. Resuspend the worms in 10 ml of M9 buffer
and inoculate into the flasks containing the resuspended bacteria. Shake the cultures at 20°C at 230
rpm.
Days 11-13: Follow the growth of the cultures carefully and harvest when majority of worms are
adults. The cultures will take about 3-3.5 days depending on the age of the worms on the plates
you inoculate with—and the desired state of the final culture. If needed, you can adjust the
temperature (16°C – 22°C) to speed up or slow down the growth
Assaying Cultures:
1. Pipet 5 ml from 2 separate flasks into 2 15 ml conicals (do in hood)
2. Spin in tabletop 600 g for 3 min with DECEL at 3
3. Aspirate sup and resuspend worms in 15 ml M9 and repellet
4. Aspirate M9 and using cutoff tip pipet worm slurry onto a glass slide and look at the worms
Harvesting Large Scale Cultures & Embryo Isolation:
1. Transfer each culture into a 500 ml graduated cylinder (rinsed with 70% EtOH and then sterile
H20) and settle worms in coldroom for 4 hrs to overnight.
2. Aspirate media using a cutoff sterile 5 ml pipet and transfer slurry from each cylinder to a 50 ml
conical
3. Pellet in megafuge at 600 g for 3’ with deceleration set to 3
4. Aspirate sup, collect two tubes into a single 50 ml conical and wash 1X with ice-cold M9 as above.
5. Resuspend in M9 to25 ml and add 25 ml cold 60% Sucrose. Mix and pellet at 1500 g for 5 min.
Adult worms should be at the top of the tube (can check bottom crude on a slide).
6. Remove adults down to 35 ml mark into a fresh 50 ml conical. Wash 1x with M9.
7. Add 25 ml 0.1 M NaCl and mix by pipetting up and down twice. Incubate on ice for 5 min.
8. Aspirate supernatant including worms that have not settled to the bottom and add 0.1 M NaCl up
to a volume of 30 ml.
9. Mix 5 ml 5 N NaOH with 10 ml bleach (use immediately). Add NaOH/Bleach mix to 30 ml worm
suspension.
10. Vortex at setting 10 for 10 s and stand tube at room temperature for 2 min. Repeat 4 times for a
total bleaching time of 10 min.
Note: Some strains are very sensitive to bleach. Watch color – bleached worms will turn a burnt
orange. If this occurs early, you can check culture on a slide and pellet before 10 min.
11. Immediately centrifuge at 800 g for 1 min at 4°C.
12. Add sterile water to a total volume of 30 ml. Vortex for 5 s and add water to 50 ml. Centrifuge at
700 g for 2.
13. Repeat step 12.
14. Add 55 ml M9 and transfer to a 500 ml flask. Shake at 22°C until embryos hatch (~24 hrs).
Seeding New Cultures Using Starved L1s:
2 Days Before: Start 50 ml culture of OP50-1 in LB + 50 µg/ml streptomycin
Day Before: Start 4 x 1.5 L cultures of OP50-1 in LB + 50 µg/ml streptomycin
Day of:
1. Harvest bacterial cultures at 4200 rpm for 15’ with decel set at 4 in sterile 1 L bottles. Pour off LB.
2. Make S Complete by adding trace metals, potassium citrate and divalent cation stocks to S-basal.
3. Resuspend bacterial pellets in 20 ml S Complete, vortext to resuspend, and transfer into sterile
2.8L fernbachs
4. After flasks with bacterial food are ready, start processing L1s. In the hood, transfer L1s to a 50 ml
conical.
5. Wash 2 x 50 ml sterile M9 (600 g for 3’)--. This removes dauer pheremone.
6. Transfer to 15 ml conical and pellet; estimate volume of pellet using a separate conical, adding
known amounts of water and comparing to pellet.
7. Resuspend to total volume of 12 ml using sterile M9 and look at sample under dissection scope;
estimate %L1s and %other crap (dead embryos/worm parts/clumps)
8. Seed each flask with equivalent of 50 µl pure L1 pellet (e.g. if pellet volume is 0.6 ml and % L1 in
the resuspension is 70% then seed with 1.4 ml of the resuspended pellet)
Try to avoid overseeding or cultures will starve!
9. Put flasks at 20°C at 230 rpm. Grow for 48 hrs and check culture. Shift down to 16-18°C
(depending on age) and harvest the next morning (~66-68 hrs total). Can adjust growth time by
shifting temperature (16°-22°C).
Freezing Adults for Purifications
1. Take cultures with synchronized adults that have just begun to have visible embryos (i.e., not old
adults but a nice string of ~10 embryos).
2. Pour into 1 L bottles. Place on ice for ~30 min. to stop worms moving. Pellet in 1 L bottles in
9.100 rotor at 700 g for 6 minutes with brake on slow.
3. Remove media until ~100-200 ml remains without disturbing worms. Move contents to 50 ml
conicals and repellet at 600 g for 3 min. in a tabletop centrifuge.
4. Wash with 50 ml cold M9. At this step, can combine ~4 conicals into a single tube.
5. Add 25 ml cold M9 and 25 ml cold 60% sucrose and mix. Depending on size of worm pellet, can
do 1-3 sucrose cushions. Pellet at 1500 g for 5 minutes.
6. Remove worms from the top of tube (down to 35 ml mark) and wash with M9.
7. Wash with 50 ml cold 1x lysis buffer (without NP40).
8. Remove lysis buffer until only a small amount remains. Drop freeze adults directly in liquid N2.
Worms will form small beads as they enter the liquid N2. Add a small volume of additional lysis
buffer to rinse out remaining worms. Beads can be harvested using a fine metal strainer (like you
would use for pasta) and stored at –80°C in a falcoln tube.
9. Grind worms with a mortar and pestle using liquid N2. Pre-chill the mortar and pestle for at least
5 minutes with liquid N2 prior to grinding. Add cells to mortar and grind for ~1 minute. Add
additional liquid N2 and scrape down cells to middle of mortar. Repeat 2-3x. Ground cells can be
stored at –80°C indefinitely.
10. Grind using a mortar and pestle (for ~5 g) or a Warring blender (40 + g) with liquid N2.
III. Coupling GFP antibody to Protein A Beads:
-Amounts below are for 1 tube of antibody
-For 1 TAP prep, prepare 4 tubes
-Volume is for settled beads not for slurry
1. Equilibrate ~110 µl Affi-Prep Protein A beads (Biorad cat# 156-0006) into PBST (PBS + 0.1%
Tween-20) in an eppendorf tube. Wash 3-4 X 1 ml; use minifuge to pellet between washes (~10-15
sec).
2. Resuspend beads in 500 µl PBST and add 55 µg of affinity-purified GFP antibody. Mix for 30 min.
- 1hr at RT.
Note: For our purifications, we generated a polyclonal antibody through Covance Research Products.
Affinity-purified antibodies are available from a variety of sources including Covance (#MMS118P; $495/1 mg), Santa Cruz Biotechnology (#sc-8334; $230/200 µg), and Research Diagnostics
(#RDI-GRNFP3abm; $500/1 mg). In addition, pre-coupled antibodies are available from Santa
Cruz Biotechnology (sc-8334AC; $265 for 500 µg/0.25 ml agarose).
3. During incubation, remove DMP from the freezer and let bottle sit tightly closed at RT for 20 min
before opening.
4. Wash beads 3X with 1 ml PBST.
5. Wash beads 3X with 1 ml 0.2M sodium borate, pH 9.0 (dilute from a stock of 1M sodium borate,
pH 9.0; make the stock from boric acid by pHing with NaOH). After the final wash, add 900 µl of
the 0.2M sodium borate, pH 9 to bring the final volume to ~ 1ml.
6. Add 100 µl of 220 mM dimethylpimelimidate (DMP; 20 mM final; Sigma D 8388; FW 259.2; stored
in a dessicated box at –20° C). Rotate tubes gently at RT for 30 min.
To make the DMP: Weigh out DMP and leave dry until just before use. Resuspend in
appropriate volume of 0.2M sodium borate, pH 9 and add immediately to the bead suspension
(e.g. for 34 mg DMP add 596 µl sodium borate).
7. After incubation with DMP, wash beads 2X with 1 ml 0.2M ethanolamine, 0.2M NaCl pH 8.5 to
inactivate the residual crosslinker. Resuspend in 1ml of the same buffer and rotate for 1h at RT.
Resuspend beads in 500 µl. You can leave the beads in 0.2 M ethanolamine, 0.2M NaCl pH 8.5 at
4° C until use. Beads are stable for several months at 4° C.
IV. Making Worm Extract
1. Weigh out 4.5 – 5 g frozen ground adults and add equal volume of 1.5 x Lysis Buffer (plus
protease inhibitors; 1 Roche tablet, 1 mM PMSF, and 1 mM Benzamidine). Thaw in 37°C water
bath until mostly melted.
2. Setup sonication ice-water bath and sonicate
- 30% amplitude for 3 min total (15 s on; 45 s off - after each 1 min wait ~2 min to chill)
- 40% amplitude for 30s (15 s on; 45 s off)
3. Transfer crude extract to TLA100.3 tube and spin at 20,000 rpm for 10 min at 2° C, DECEL = 5
4. Remove sup and pellet at 50,000 rpm for 20 min at 2° C. Try to avoid any lipid and repellet if too
cloudy.
5. Collect sup into a tube on ice. Add KCl to a final concentration of 300 mM.
V. GFP Immunoprecipitation
• For a standard TAP prep, use 4 tubes of coupled antibody
1. Pre-elute 100 µl of coupled beads (equivalent to 1 tube from Part II) 3X with 1 ml of 100 mM
glycine pH 2.5; this gets rid of uncoupled/elutable antibodies and reduces background. Do this
quickly – don’t leave beads for long time in glycine.
2. Wash beads 3 x 1ml into Lysis Buffer (with 300 mM KCl) to neutralize glycine. On last wash, split
100 µl of beads into two separate tubes. At this stage for a TAP prep, you will have 8 tubes.
3. Mix each tube of beads with ~1 ml HSS extract for 1 hr at 4° C.
4. Rinse beads 3X with 1 ml Lysis Buffer containing 300 mM KCl, 0.05% NP-40, 0.5 mM DTT, and
protease inhibitors (1:1000 LPC; 1:2000 E-64)
5. Wash beads 2 x 5 min with 1 ml Lysis Buffer containing 300 mM KCl, 0.05% NP-40, 0.5 mM DTT,
and protease inhibitors (1:1000 LPC; 1:2000 E-64). Do one wash without protease inhibitors before
adding the TEV.
VI. TEV Cleavage
1. Pool 4 tubes of GFP antibody beads into a single tube and fill with Lysis buffer containing 300 mM
KCl, 0.05% NP-40, 0.5 mM DTT (2 tubes total).
2. Add ~40 µl of Nickel/FPLC purified stable TEV protease (~1 mg/ml stock; see Kapust et al.
(2001). Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants
with wild-type catalytic proficiency. Protein Eng. 14:993-1000) and rock tubes overnight at 4° C.
(Also available from Invitrogen – cat # 12575-015).
3. Pellet tubes and remove supernatant to a fresh tube. Add an additional 350 µl Lysis buffer to
beads to remove any residual protein.
VII. S Protein Agarose
1. Wash 2 tubes of 80 µl S protein agarose slurry (40 µl packed resin; Novagen cat# 69704) with 3 x 1
ml Lysis buffer (300 mM KCl).
2. Add TEV eluted supernatant to S protein agarose and rock for 3 hrs at 4°C.
3. Pellet beads and wash 3 x with Lysis buffer (with 300 mM KCl)
4. Wash 1x with Lysis Buffer with 100 mM KCl without detergent.
VIII. Urea Elution
1. Wash beads with pre-urea wash buffer (50 mM Tris pH 8.5, 1 mM EGTA, 75 mM KCl) Transfer to
a 0.5 ml ependorf tube. Remove all residual supernatant.
2. Add 65 µl Urea elution buffer (50 mM Tris pH 8.5, 8 M Urea – For urea, use Invitrogen cat #
15505-035 and make up urea fresh every time) and rotate for 30 min at RT.
3. Pellet beads and remove 50 µl urea to a new 0.5 ml tube. Drop freeze in liquid N2.
4. Add ~20 µl 3 x sample buffer to the remaining urea and S protein agarose. Boil for 3 min.
- For a gel sample, you can also add sample buffer to the S protein agarose without first doing the
Urea elution.
Solutions and Media:
Lysis Buffer: (H100 + 10% glycerol)
50 mM HEPES, pH 7.4
1 mM EGTA
1 mM MgCl2
100 mM KCl
10% glycerol
0.05% NP-40
- Just prior to use, to 5 ml lysis buffer, add 1 tablet mini EDTA-free Complete PI tablet (Roche cat#
1 836 170)
S basal (per L):
5.9 g NaCl
50 ml of 1M potassium phosphate pH 6.0
1 ml of cholesterol (5 mg/ml in EtOH)
- Split into 2 x 500 ml in 2.8 L ferbach flasks; to each flask add 0.5 ml 5 mg/ml cholesterol (in
EtOH; should form a light cloudy precipitate)
Autoclave and store at RT
Complete S Basal:
To each 500 ml add:
5 ml 1M Potassium citrate, pH 6.0
5 ml Trace Metals solution
1.5 ml 1M MgSO4
1.5 ml 1M CaCl2
Trace Metals Solution:
Disodium EDTA
FeSO4 7H2O
MnCl2 4H2O
ZnSO4 7H2O
CuSO4 5H2O
1.86 g
0.69 g
0.20 g
0.29 g
0.025g
(5 mM)
(2.5 mM)
(1 mM)
(1 mM)
(0.1 mM)
Dissolve in 1L water; aliquot into 50 ml conicals and store in dark
1M Potassium Citrate, pH 6.0
210.1 g citric acid monohydrate; water to 900 ml
Adjust pH to 6.0 using 10N KOH (or pellets) and bring up to 1L
Autoclave and store at RT