Worm Lysate Preparation & Western Blot Analysis

Updated by Christian Nelson June 2016

Worm Lysate Prep
1. Grow worms of desired genotypes on 10 NGM+OP50 plates (can pick or chunk) until nearly starved.

2. Wash worms from plates (M9 + 0.1% TX-100) into single 15mL conical tube.

3. Spin worms 2 minutes at 2000rpm in table top centrifuge to pellet.

4. Remove sup.

5. Resuspend worms in 500uL Buffer H supplemented with protease inhibitors.

6. Transfer to capped microfuge tube and keep lysates on ice from here forward.

7. Add one cap full of glass beads (Biospec).

8. Bead beat 3x30sec with 1min rest between sessions in cold room.

9. Sonicate in water bath sonicator 2x30sec on medium setting.

10. Spin 10min at max speed (14k) in cold room microfuge.

11. Remove sup to new tube (~400uL).

12. Determine protein concentration using Bradford Assay on spectrophotometer.

13. Add ½ volume 4X Sample Buffer (e.g. 200uL).

14. Boil samples for 5 minutes prior to loading on SDS-PAGE gel.

Western Blotting
1. Prepare SDS-PAGE gel and load samples. See chart on website for SDS-PAGE preparation help.

2. Run gel at 30mA for ~2 hours or until dye front runs off into buffer.

3. Do Semi-Dry transfer using Bio-Rad Trans Blot Turbo system (see their protocol) using their reagents.

4. Briefly rinse blot (nitrocellulose) in MilliQ water, ponceau stain for ~30 seconds, and wash with MilliQ water. This will provide an indication of your lysate quality, transfer success, and whether you loaded equivalent amounts of protein between lanes.

5. Block for 30min at RT in PBST + 5% w/v non-fat milk solution.

6. Incubate in PBST + primary antibody overnight in cold room on rocker.

7. Wash 3X in PBST for 10min at RT.

8. Incubate in PBST + secondary antibody (mouse/rabbit HRP conjugate) for 1 hour at RT.

9. Wash 3X in PBST for 10min at RT.

10. Probe blot with ECL solution (Pico ECL for strong signal, Dura ECL for weak signal) for 1 min, then expose to film in dark room.

Reagents
Buffer H: A large volume of this can be made and kept indefinitely at 4C.
 * 50mM HEPES pH8.0
 * 2mM MgCl2
 * 0.1mM EDTA pH8.0
 * 0.5mM EGTA-KOH pH8.0
 * 15% Glycerol
 * 0.1% NP-40
 * 500mM KCl

Protease inhibitors Sample buffer: Ponceau Stain (1L):
 * complete mini tablets w/o EGTA [Roche]
 * 0.1 mM AEBSF
 * 5 mM benzamidine
 * 10ug/mL aprotinin
 * 160mM Tris pH6.8
 * 4% SDS, 20% glycerol
 * 20mM EDTA
 * .0026% bromophenol blue.
 * 1gram Ponceau Stain
 * 50mL acetic acid
 * water to 1L.

Common Antibodies
1. Anti-FLAG M2, Mouse, Sigma Aldrich Product #F1804, 1:5000

2. Anti-GFP Living Colors, Rabbit, Clontech Product #632592, 1:1000

3. Anti-HA 12CA5, Mouse, Santa Cruz Biotech Product #SC575-92, 1:1000

4. Anti-Tubulin DM1A, Mouse, Sigma Aldrich Product #T9026, 1:3500

5. Anti-GAPDH 6C5, Mouse, Calbiochem Product #CB1001, 1:1000