Worm Breeder's Gazette 12(3): 14 (June 15, 1992)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Non-Radioactive Labeling of C. elegans Protein Extracts for Immunoprecipitations

Sonya Gettner, Cynthia Kenyon, Louis Reichardt

Department of physiology, Box 0444 UCSF, San Francisco, CA 94143

We have recently labelled protein extracts prepared from C. elegans with suHo-NHS-biotin (Pierce, cat. #21217) for immunoprecipitations as an alternative to metabolically labeling worms with 35S-methionine and 35S-cysteine. For membrane proteins, the unreacted biotin is separated from the protein extract after labeling by using a high speed spin to pellet the membrane fractions. In addition, excess Tris is added to stop the reaction. After immunoprecipitation, the proteins are run on SDS-PAGE and transferred to nitrocellulose. The nitrocellulose is processed as a Western blot, using alkaline phosphatase-conjugated streptavidin (Accurate) as the secondary reagent. We have detected immunoprecipitates of the integrin ß subunit with its three associated a subunits with alkaline phosphatase from as little as 10 µl of worms. Visualization using the ECL kit from Amersham could potentially increase the sensitivity up to tenfold.

Although this procedure has been worked out for membrane proteins, it should be possible to modify it for soluble proteins. Again, to stop the labelling reaction excess Tris-CI can be added and the reaction time extended as the sulfo-NHS-biotin has a half-life of hydrolysis of 1-2 hours in aqueous solution.

The immunoprecipitation procedure is fairly lengthy so we are including here only the procedure for preparing and labeling the protein extract. Call (1415-476-1034)or fax us (1415-566-4969) if you are interested in the procedure we have used for immunoprecipitations. A standard reference is Miyake et al. 1991. J.Exp. Med. 173, 599-607.

1 ) Prepare Protein extract

-Pellets of worms (cleaned by floating on a sucrose gradient and washed in M9 )can be frozen in a dry ice/ethanol bath and stored at -20 °C. (0.5 ml is what is used in this procedure)

-Resuspend worms in 4X volume of ice cold lysis buffer (0.1 M HEPES, pH 8.0 in PBS-; 0.32 M sucrose). Add 2X volume of sand (Sigma, white quartz) and vortex with 1 minute bursts separated by 30 second intervals on ice for a total of 15 minutes. Extract can be checked under a dissecting scope for empty cuticles to determine effectiveness of Iysis.

-Spin on setting 3 in a clinical centrifuge for 1 minute to remove sand. Transfer supernatant to two 1.5 ml eppendorf tubes; spin 5,000 rpm in a refrigerated microfuge for 5 minutes to remove nuclei and cytoskeleton .

-Transfer supernatants to two fresh eppendorf tubes and spin 15,000 rpm at 4 °C for 30 minutes to pellet membranes (see notes above for soluble proteins). Remove supernatants and resuspend pellets in 1 ml total biotinylation buffer (0.1 M HEPES, pH 8.0 in PBS).

2) Biotinylation of protein extract

-Add 20 µl sulfo-NHS-biotin to the protein extract. Incubate at 4 °C for 2 hours with rocking.

-To stop reaction, add 100 µl of 1 M Tris-CI, pH 8.0.

-Spin 15,000 rpm for 20 minutes at 4 °C to pellet membranes. Wash membranes 3X with 0.1 M Tris-CI, pH 8.0, spinning at 15,000 rpm for 20 minutes at 4 °C in between each wash.

-Resuspend pellet in 1 ml ice cold immunoprecipitation buffer after final spin.

·PBS: (0.1 g/L C aCl2 ,0.1 g/L M gCl2 ,0.2 g/L KCl, 0.2 g/L KH2 PO4,8 g/L NaCI, 2.16 g/L N a2 HPO47H20)

[Figure not included; not readable]