Preparation of Worm Extracts by Sonication
By Sudhir Nayak (Schedl Lab)
This protocol was designed to rapidly generate small scale cytosolic extracts of C. elegans for Western or IP (has not been tested for RNA work). The protocol works well for between 50 to 5000 worms and has not been extensively tested on larger a scale, though it should work. All the usual caveats for working with proteins apply; keep everything cold with good sterile technique, use protease inhibitors, and your chances of success are dependent on the abundance/stability of your protein. The total time from worm to working lysate is usually less than 30min.
Solutions (COLD!):
M9 salts or PBS
ddH20
HB (homogenization buffer) see recipe
Collection
1) Either pick the desired worms into a 1.5ml tube PBS or wash them from a plate with 1.5ml PBS. Spin 2k, 1min to pellet. Keep them on ice at all times.
2) Wash the worms with PBS 2x.
3) Wash the worms with ddH20 2x.
4) Resuspend the worms in at least 250ul of HB (500ul works better). The worms are now ready for sonication.
If you want to reduce bacterial contamination wash worms for 5min each in PBS and ddH2O or perform sucrose floatation.
Sonication
1) In an ice bucket take your worms to the sonicator. The micro tip should already be attached. Make sure you throughly clean the micro tip and are familiar with the use of the sonicator before you proceed.
2) Raise the sonicator so that you can fit your ice bucket under the micro tip and have access to your 1.5ml tube.
3) Insert the micro tip into your 1.5ml tube by lifting up the ice bucket, put on the head phones, and pulse the worms for 1sec at the micro top limit (indicated on the controller) then rest on ice 30sec. Using our sonicator, 5 pulses total for 1 second each with a 30sec chill between each works well.
If you are using a different sonicator you need to determine this empirically by checking a small aliquot of your sample after each pulse. Lysis is complete when the vast majority of worms are blown apart. You have gone too far (broken up the nuclei) if the lysate becomes viscous or foams excessively. Its better to use more pulses if needed rather than increase the time of each pulse as heat is generated over time.
Clearing lysate
1) To clear lysate spin the sonicated worms at 14k for 10min at 4deg.
2) Transfer the supernatant to a fresh tube. It is not ready for IP.
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.
Recipe
HB (homogenization buffer)
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 %
Add right before use:
1mM DTT 1000x of 1M
Your favorite protease inhibitors. We use the protease inhibitors tablet from Boehringer Manheim (1836 153). One tablet per 10ml HB and tablets can be cut into smaller pieces if desired.