Co-Immunoprecipitation of Germline Proteins

*from adult C. elegans August 2014 Christian’s Method*

## **Part 1**: Grow worms & make popcorn (Dustin’s protocol w/ modifications)

* 1. Make worm popcorn from synchronized adults
* 1.1. Day 1 Grow up worms to synchronize
* 1.1.1. Add 25mL of HB101 bacteria to 1 liter of complete S media.
* 1.1.2. Wash worms off of 40 recently starved plates and add to the liter of S media and bacteria.
* 1.1.3. Divide S media + bacteria + worms into 4x 250mL glass flasks. Shake at 220rpm at 20C for 3 days.
* 1.2. Day 4 late: Harvest embryos and hatch on unseeded plates
* 1.2.1. Pour worms into 1 liter pear funnel and let settle.
* 1.2.2. Drip into 50ml conical tube.
* 1.2.3. Wash with water 3X
* 1.2.4. Throw worms into bleach solution. (~3 minutes then rinse 3X with M9)
* 1.2.4.1. 3 parts water, 1 part bleach, 0.1 part 10M NaOH (50ml – 36mL water, 12.6mL bleach, 1.4mL 10N NaOH)
* 1.2.5. Let embryos hatch on about 20 unseeded (no food) plates.
* 1.3. Day 5 mid-day: Feed L1’s
* 1.3.1. Add 40mL of HB101 bacteria to 1 liter of complete S media.
* 1.3.2. Wash hatched L1s and add to the liter of S media and bacteria
* 1.3.3. Divide S media + bacteria + worms into 4x 250mL glass flasks. Shake at 220rpm at 20C for 24 hours
* 1.4. Day 6 early: Reduce temp
* 1.4.1 Reduce temp from 20C to 18C.
* 1.4.2 Otherwise worms will be too old by harvesting time on Day 8.
* 1.5. Day 8 early Harvest adults and make popcorn
* 1.5.1. Inspect worms on dissection scope to make sure most are adults (around 68 hours after feeding L1s).
* 1.5.2. Pour worms into 1 liter pear funnel and let settle
* 1.5.3. Pre-chill 50ml conical tubes in dry ice
* 1.5.4. Collect and wash 2X in water.
* 1.5.5. Wash 1X in Buffer H (lysis buffer) with NO detergent!! (see part 3 for recipe)
* 1.5.6. Pour liquid nitrogen into 50ml pre-chilled tube
* 1.5.7. Put pelleted worms in syringe and drip worms into liquid nitrogen
* 1.5.8. Put worm popcorn in 50ml conical tubes and freeze in -80C

This protocol usually nets somewhere around 10-20mL of packed popcorn depending on how healthy your strain is and the # of adults you have at bleaching stage. I’ve had terrible luck using worms from NGM plates and ALWAYS do liquid cultures now

## **Part 2**: Make worm powder

* 2.1. Pulverize worms in Retsch Mixer Mill MM-400 under cryogenic conditions
* 2.1.1. Chill 2x 25mL grinding jars w/ balls inside in liquid nitrogen until bubbling stops
* 2.1.2. Transfer around ~15mL of popcorn to each tube, leaving room for ball to move around. DO NOT OVERFILL.
* 2.1.3. NOTES: You must ALWAYS use both jars, similar to balancing centrifuge. Both jars MUST have popcorn in them, ball will damage an empty jar.
* 2.1.4. After filled with popcorn, return ball to jar, seal, and immerse in liquid nitrogen.
* 2.1.5. Keep jars in liquid nitrogen when not milling.
* 2.1.6. Pulverize worms for 2min @ 25Hz, 3 times. Chill on liquid nitrogen between milling sessions until bubbling stops.
* 2.1.7. Carefully remove powder from jars into small box or conical tube.
* 2.1.8. Store at -80C until ready to perform IP (can be stored indefinitely).

This usually produces somewhere in the ~5g range of powder, enough for a few IPs.

## **Part 3**: Conjugate dynabeads to desired antibody (Bungo’s protocol)

* I always cross-link my antibodies to Invitrogen Dynabeads. This allows you to use the same antibody for Western blot without having to worry about heavy & light chains showing up. For a single Co-IP, I make 100uL of beads (50uL each for sample/negative control) w/ 25ug of antibody (12.5ug/IP).
* Use Protein A Dynabeads for rabbit antibodies and Protein G for mouse antibodies.
* Working IP antibodies:
	+ mouse GFP (Roche, 11814460001)
	+ mouse FLAG M2 (Sigma, F3165)
	+ rabbit A-14 Myc (Santa Cruz Biotech, SC-789)
* 3.1. Bind 25ug of antibody to 100uL of Dynabeads.
* 3.1.1. Resuspend 100uL slurry of Dynabeads.
* 3.1.2. Concentrate in magnet, remove sup.
* 3.1.3. Wash twice in 1mL of 0.1M Na-phosphate buffer pH 7.0
* 3.1.3.2. 500mL 0.1M Na phosphate pH 7.0 (58mL 0.5M Na2HPO4, 21mL 1M NaH2PO4, 421mL ddH2O)
* 3.1.4. Add 25ug of antibodies and same volume of 0.1M Na phosphate buffer.
* 3.1.4.2. e.g. if 25ug of FLAG M2 is 25uL, add 25uL of 0.1M Na phosphate
* 3.1.5. Shake with gentle agitation on vortex machine for 25min.
* 3.2. Crosslink antibody to beads
* 3.2.1. Concentrate beads, remove sup.
* 3.2.2 Wash twice in 1ml of 0.1M Na-phosphate buffer pH 7.0 with 0.01% Tween20 (Tween helps with protein stability in storage of Dynabeads)
* 3.2.3. Wash twice with 1ml of 0.2M triethanolamine, pH 8.2 (Dynabeads now have immobilized antibody)
* 3.2.4. Resuspend Dynabeads in 1ml of 20mM DMP (in common –20o C freezer, Dimethyl Pimelimidate, D8388, Sigma) in 0.2M triethanolamine, pH 8.2 (note that this crosslinking solution must be prepared immediately before use)
* 3.2.4.2. Use 5.18mg DMP in 1mL 0.2M triethanolamine
* 3.2.5. Incubuate on rotation mixer for 30 minutes at RT (DO NOT OVER CROSSLINK)
* 3.3. Stop the crosslinking reaction
* 3.3.1. Concentrate beads, remove sup.
* 3.3.2. Resuspend beads in 1mL of 50mM Tris-HCl pH=7.5
* 3.3.3. Incubate with rotation mixing for 15min at RT.
* 3.3.4. Wash beads 3X in 1mL PBST (0.1% Tween-20)
* 3.3.4 Store beads in original volume but w/ PBST (e.g. 100uL PBST) at 4C. Do not freeze.
* 3.3.5 Beads can be stored for around ~1 month at 4C without any reduction in efficiency. No need to equilibrate prior to use.

## **Part 4**: Lyse Worms & perform IP

* Lysis buffer (Buffer H):
	+ 50mM Hepes pH 8.0
	+ 150mM KCl, 2mM MgCl2
	+ 15% Glycerol
	+ 0.1mM EDTA pH 8.0
	+ 0.5mM EGTA-KOH pH 8.0
	+ 0.1% NP-40
* Protease inhibitors (working concentrations):
	+ 0.1 mM AEBSF
	+ 5 mM benzamidine
	+ 1:200 aprotinin
	+ Roche® Complete Mini tablets w/o EGTA
* Phosphatase inhibitors (working concentrations):
	+ 1mM Na4P2O7
	+ 2mM Na-β-glcyerophosphate
	+ 0.1mM Na3VO4
	+ 5mM NaF
* 2X Sample Buffer (2XSB):
	+ 4% SDS
	+ 20% Glycerol
	+ 120mM Tric-HCl pH 6.8
	+ 0.02% bromophenol blue (add 2-mercaptoethanol [B-Me] to 5% prior to use)
* 4.1. Lyse worms
* 4.1.1. Weigh 1g of worm powder for each strain you plan to use into 15mL conical tube (always do untagged negative control).
* 4.1.2. Resuspend in 5mL ice cold Buffer H w/ protease + phosphatase inhibitors.
* 4.1.3. Rotate 3min @ 4C or until re-suspended well.
* 4.1.4. Sonicate 2x30sec at medium amplitude with 1min rest on ice.
* 4.1.5. Spin @ ~50,000xg (max speed) @4C for 20’ in JA-20 rotor.
* 4.1.5.2. Can do optional ~15min ultracentrifuge spin @ 4C. This helps get rid of lipids and seems to help with IP, but not necessary.
* 4.1.6. Take sup, move to new 15mL conical avoiding top lipid layer.
* 4.2. Perform IP
* 4.2.1. Take 50uL ‘input’ sample for each strain, add 50uL 2X SB+B-Me, boil 3 min, store at -20C.
* 4.2.2. Add 50uL of Dynabeads with conjugated antibody.
* 4.2.2.2. Note this is usually not enough to immuno-deplete, could increase to get more protein.
* 4.2.3. Rotate at 4C for 2-3hours.
* 4.2.4. Concentrate beads in magnet.
* 4.2.5. Take 50uL of sup as ‘unbound’ sample for each strain, add 50uL 2X SB+BMe, boil 3 min, store at -20C. 4.2.6. Remove sup, wash beads 4X with 1mL ice cold Buffer H.
* 4.2.7. Remove as much sup as possible.
* 4.2.8. Boil in 50uL 2X SB+B-Me to elute IP’d proteins.
* 4.2.9. Concentrate in magnet, take sup (eluate) to new tube (removing beads).

## **Part 5**: Analyze IP’d proteins by Western blot

* See Western protocol.
* I usually run 10% of IP sample (5uL) to analyze for the presence of IP’d protein (e.g. protein with GFP/FLAG tag)
* To analyze for presence of Co-IP’d proteins, I run significantly more, up to 50% (25uL) of IP sample, depending on stoichiometry of complex.
* For inputs/unbound samples, I run 10uL each for Western.