co-Immunoprecipitation

 

A modified protocol for co-immunoprecipitation based on that presented in Goodwin, E. B., Okkema, P. G., Evans, T. C. & Kimble, J. Translational regulation of tra-2 by its 3' untranslated region controls sexual identity in C. elegans. Cell 75, 329-39 (1993).

 

Homogenization Buffer (HB)

 

150 mM NaCl 30 mls of 5.0 M

50 mM Hepes pH 7.6 100 mls of 0.5 M

10 mM MgCl2 10 mls of 1.0 M

1 mM EGTA 10 mls of 0.1 M

15 mM EDTA 30 mls of 0.5 M

0.6 mg/ml Heparin 130 mgs

10% Glycerol 100 mls

 

DEPC treat the solution, then autoclave.

Before using, add the following:

1 mM DTT 1000X of 1M

8 mM vanadyl ribonucleoside complex 25X of 200 mM solution

400 U RNasin/ 8ml

protease inhibitor cocktail tablet

 

Lysis of the Worms

1.       Two days after harvesting embryos, feed the samples for three hours with ~1.5 mls of 3X diluted packed E coli.

 

2.       Collect the worms, rinse them twice and resuspend them in 30 mls of M9 and add 400 ul of formaldehyde for 60 minutes at 4C.

3.       Before French pressing all samples, rinse them once in HB buffer. Resuspend the worms in 3 volumes of new HB.

 

4.       French pressed all samples 2-3 times at 6000 PSI in a small cell, then wheaton homogenize them with 30 passes. During homogenization, add ~0.6 ml of HB to each sample to rinse sample off of the glass.

 

5.       Spin down debris using a J-20 rotor at 5000 rpm for 6 minutes

 

6.       Spin down the supernatant using a J-20 rotor at 14000 rpm for 20 minutes.

 

7.       At this point, you can flash freeze the samples.

The IP

1. I do the IP at 4C for ~2.0 hours. Each sample is done in 1.8 ml tubes.

Sample vol 1 ml

Rnasin 10 ul

Vanadyl Ribonucleoside Complex

M2 Beads 100 ul

(Note- the M2 Beads are Anti-Flag AB-conjugated beads from Sigma)

 

2. Wash the mRNA/FLAG::PABP/bead complexes in HB 4 times on ice.

 

3. Resuspend the beads in 100 ul of 1X IP elution buffer (50 mM Tris/HCl pH8.0, 10mM EDTA, 1.3 %SDS). Add about 40 U of Rnasin to each tube. Incubate at 65C for 30 minutes. Gently spin the beads down, remove the supernatent and store in a tube on ice. Add another 100 ul of 1X IP elution buffer to the beads and repeat. Combine the two eluates.

 

4. Add 4X vol trisol to the eluate. Let stand for 5 min at RT. Mix and add1 vol of Chl. Let stand for 10 min. Spin for 15 minutes at 4C.

 

5. Extract with Chl (x1), then add 500 ul of iso-propyl, incubate at RT for 10 minutes and spun for 25 minutes at 4C. Rinse twice with 1 ml of 70% ETOH.

 

6. Take the OD readings.