PROTOCOLS       

Miyuki's IP Protocol

• 10 cm Egg plate x 2~3
• Wash until most E. coli and debris are gone.
• Divide 0.3 mL ~ 0.5 mL worm pellet
• Add 3~4 mL lysis B (Adjust volume B so that worm/B ratio of two samples becomes similar.)
• Lyse in grinder pre-chilled on ice. (Check by microscope every 3~5 strokes. I did not do too much. I stopped when 50-70% of the worms were lysed.)
• Incubate lysate on ice for 30 minutes.
• Centrifuge at 12,000 rpm for 5 minutes.
• Collect the supernatant.
• Divide the supernatant into about 10 aliquots and keep some for total lysate control.
• Add antibodies.
• Put at 4 °C for 2 hours or 1 hour.
• Add protein-A sephorose (50%) ~ 20 mL
• Put at 4 °C for 1 hour.
• Wash 3 times.
• Take all buffer out.
• Add 1x Sampling buffer with b mercaptoethanol.
• Boil for 5 minutes.
• Perform Western.
• For one tube:
  α-GFP 3E6 (3mL was good enough for GFP::RME-6)
  α-GFP rabbit polyclonal (3mL)
• IP efficiency check
  3~5% of last eluent
  3-5% of starting whole lysate in 1 tube & compare w/ above

How much whole lysate you need for 1 tube and how much antibody is enough depends upon the expression level and antibody. Ideally, you should do a test experiment to check the efficiency or a comparison to a known sample like GFP::RME-6.

Immunoprecipitation from whole worm lysates

Transgenic worms expressing GFP::RME-6 (pwIs2) in the rme-6(b1014) background were used for coimmunoprecipitation analysis. Approximate 0.3 ml of young adults were harvested and washed with M9 buffer. They were resuspendend in 25 mM Hepes-KOH [pH7.4], 125 mM potassium acetate, 5 mM magnesium acetate, 1 mM dithiothreitol, 10% glycerol (IP buffer) containing 0.5% NP-40 and protease inhibitors and homogenized in a stainless steel homogenizer (Wheaton). Homogenates were incubated on ice for 30min and cleared by centrifugation for 5 min at 16,000 x g at 4°C. For immunoprecipitation, extracts were incubated with anti-GFP monoclonal antibody (3E6) for 1 h followed by incubation with protein A-Sepharose (Sigma) for 1 h. Beads were washed with IP buffer containing 0.1% NP-40 three times. Precipitants were subjected to immunoblotting using anti-α-adaptin and anti-EEA-1 antibodies. For a reverse experiment, α-adaptin was immunoprecipitated with anti-α-adaptin antibody from worm extracts, and precipitants were examined by immunoblotting using goat anti-GFP antibody. Control experiments using anti-GFP antibodies on N2 lysates failed to precipitate detectable α-adaptin (data not shown).