Cytosolic Extract & Immunoprecipitation
Prepared by Min-Ho Lee and Sudhir Nayak
1. Culture
There are 2 ways we start worm culture:
EASY (but less synchronous culture): Start 100 ml liquid culture using the standard protocol from 10-20 large plates (10cm) of nearly starved N2 (lots of starved L1 on plates). When most of the worms are adults (3-4 days at 20 oC) harvest them. Do not starve them. About 80% of worms will be adults.
HARD (but more synchronous culture): Start 100 ml liquid culture using the standard protocol from 4 large plates (10cm) of nearly starved N2 (lots of starved L1 on plates). When most of the worms are adults (3-4 days at 20 oC) egg prep them and start a new 100 ml liquid culture and grow for 3-4 days at 20 oC). Do not starve them. Nearly 100% of worms will be adults.
2. Harvest
Keep everything cold and work as quickly as possible
SOLUTIONS: cold 0.1 N NaCl, cold ddH2O, cold HB (homogenization buffer), cold 60% sucrose. Solutions can be pre-made and left in the cold room.
The actual size of the rotor, centrifuge and volumes you need will depend on your application. The following is for 100ml liquid culture but can be scaled up to 1L or more.
A) Spin down worms in swinging bucket rotor, 2k rpm, 2 min (remember to keep everything cold). You can split the 100ml culture into 5x50ml conicals to give you a balance and make this protocol easier.
B) Wash 4x in 50ml cold 0.1 N NaCl. Spin 2k rpm, 2min to pellet each time.
C) Sucrose floatation. COLD!!! 20 ml (worms in 0.1N NaCl) + 20 ml 60 % sucrose, mix and spin in swinging bucket rotor 2K, 2min. Any volume of worms can be used as long as you use 30% final sucrose for the floatation. ADDITION: If the worms do not appear clean then wash them 2 times in 0.1N NaCl and repeat sucrose floatation. Each time you do a float your worm yield will decrease but you may get cleaner worms.
D) Collect floating worms and wash 2x with cold water in a 15ml conical.
E) Wash 1x in HB without DTT and Protease.
F) Add 3x volume (packed volume of worms) of HB to worm pellet. This HB should contain DTT and Protease inhibitors. The worms are now ready for extraction. From 250 ml liquid culture (3 days at 20 oC) we get 3-5 ml of packed worms.
3. Extract
Keep everything cold including the French press cell. Make sure you wash the cell with DEPC water put the cell at 4C O/N to cool completely if you are going to do RNA work. Not quite as important for protein work.
A) Pass the worms through the French Press twice at 4000 psi. This will break down worms into several pieces and the conditions (appropriate pressure and number of passes) have to be empirically determined for each press and cell. The conditions are ‘correct’ for making a germ line extract when 99% of the animals are broken into 2-4 pieces. Pressures as high as 12000psi can be used without shredding DNA and still produce good quality extract.
B) Spin down debris 1K (200 g), 2 min. This removes the majority of worm carcasses.
C) Collect sup. and transfer to dounce homogeniger (10 ml or 20ml).
D) 25 Strokes with B(or A) pestle. Avoid foaming.
E) Spin down 2K (800 g), 5 min. Removes smaller pieces, remaining nuclei, etc.
F) Collect sup. (turbid) and spin 14k (20k x G), 20 min. Clears lysate.
G) Collect sup. Add 1/10th vol. of cold 15mM Hepes pH7.6 and 1/10th vol. of cold 1M NaCl soln. You can also normalize this to your favorite buffer conditions if you don’t like Hepes/NaCl.
H) Use immediately for IP or aliquot (500ul) and keep at -80 oC (see storage).
I) Measure protein concentration (micro BCA from Pierce).
J) Check extract by Western blotting for a known protein of interest.
STORAGE: To store the extract at –80 oC supplement lysate with 15% glycerol. DO NOT FREEZE/THAW for either RNA or protein work. Its best if you don’t freeze at all if you plan on doing quantative RNA work.
Homogenization buffer (HB)
DEPC treated reagents only for RNA work.
for 500 ml
15 mM Hepes pH 7.6 15 ml of 0.5 M
10 mM KCl 2.5 ml of 2 M
1.5 mM MgCl2 0.75 ml of 1 M
0.1 mM EDTA 100 ul of 0.5 M
0.5 mM EGTA 2.5 ml of 0.1 M
44 mM Sucrose 14.7 ml of 50 %
KEEP COLD!!!
Add right before adding to worm pellet.
1mM DTT 1000x of 1M
Protease inhibitor cocktail tablet from Boehringer Manheim (1836 153). One tablet per 10ml HB.
Immunoprecipitation
A) Preparation of Protein A/G-Sepharose beads
1. Transfer 0.2 ml bed volume of protein A- or protein G- sepharose beads to a 15 ml centrifuge tube.
2. Add 10ml of ice cold PBS and centrifuge for 5 minutes at 2000xg, 4oC. Discard the supernatant. Repeat 2x.
3. Suspend beads in 0.2ml of IP buffer (Homogenization Buffer (above) + 100mM Nacl)
This same basic procedure applies to other anti-epitope tag beads like myc, HA, FLAG, GFP, etc., however, the amounts need to he refined for your particular application.
B) Perform binding of the Sepharose beads with epitope-specific antibody
1. Add 20ug of the epitope specific antibody to the sepharose beads prepared as above.
2. Shake for 1 hr (to ON) at 4oC and then wash three times with the IP Buffer.
C) Perform selection with epitope-specific antibody
1. For each immunoprecipitation use 200ul of the cytosolic extract. The protein concentration is usually at ~5-10ug/ul if made as described above. The amount of extract (total protein) that you will need depends on a variety of factors including the abundance of your protein (representation in the extract), how efficiently it was extracted, and how efficiently you can IP it from the extract.
2. Add lysate to the the sepharose beads bound to the antibody of interest.
3. Shake the mixture for 2hrs (to ON) 4oC on an orbital shaker
4. Wash the beads the next morning 4-5 times with IP buffer.
5. Add SDS-sample buffer and boil the beads (for not more than 2 minutes), and analyze on SDS-PAGE. You can also elute with excess epitope tag (eg FLAG peptide) and process the sup for SDS-PAGE.
ALTERNATE: You can add 1-10ug of antibody to your extract first and bind for 15min to 1 hr then IP with the appropriate anti-epitope tagged beads.
There are many variations to the IP protocol that are not detailed here. Inclusion of detergent (tween 0.1%) in the IP buffer or washes, addition of high salt (300-500mM NaCl) to the IP buffer or washes, etc. The conditions will vary based on your IP needs.