Worm Breeder's Gazette 7(2): 28
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.
Specific antibodies can be used both to localize proteins in cells and to identify genes coding for these proteins either by immunoprecipitation of hybrid related in vitro translations or by screening of expression libraries. We have prepared antibodies to three sperm-specific proteins by using spots cut out of 2-D gels for immunization of rabbits. We first identified those proteins that were likely to be sperm-specific by running 2-D gels of [35S]-labeled sperm together with excess cold total hermaphrodite protein and vice-versa. We then compared [35S]-labeled in vitro translated male poly A+ RNA peptides with sperm proteins. Seven major spots were found that appeared to be both sperm specific and present in male poly A+ RNA translations. Five of these spots were cut out of Coomassie blue stained 2-D gels that had been loaded with 3 x 10+E7 cells (480 g total protein). Spots from two gels were combined, broken up by passage through a 20 gauge needle, emulsified with Freund's complete adjuvant and injected subcutaneously into rabbits. At 1 month intervals, rabbits were boosted with the same antigen in incomplete Freunds. Each injection was about 70 g of the major sperm protein ( MSP) and 10 g or less of the other proteins. The sera were tested by indirect immunofluorescence staining and immunoprecipitation. Three out of 5 rabbits were positive. Anti MSP stained cells at a dilution of 1:16,000; anti 'spot 10', a 13,000 dalton sperm protein, stained at 1:16,000; anti 'spot 12', a 15,000 dalton protein, stained at 1:800. All three anti sera immunoprecipitated the injected antigen. All three stained sperm and spermatocytes and no other worm tissue the staining of spermatozoa was predominately in the pseudopod. These results show that spots off 2-D gels can provide sufficient antigen for preparing high titer antisera. The preparative gels can be stored at -70 C after soaking in 50% glycerol so additional spots can be cut out for subsequent immunizations. Technical note: For immunofluorescent staining of sperm and testes, I find that fixation with formaldehyde gives much better morphological preservation and clearer localizations than does fixation in organic solvents. I use 4% formaldehyde in PBS. The formaldehyde must be freshly prepared just before use from paraformaldehyde by heating to 90 C then treating with NaOH until the solution clears and then neutralizing with HCl. Dissected gonads are fixed on BSA or polylysine subbed slides for 20 minutes under a coverslip then the coverslip is removed after by freezing on dry ice. Unreacted aldehydes are blocked by 10 mg/ml glycine in PBS and then cells are permeabilized with 0.5% Triton X-100 for 5 minutes, rinsed, and stained as desired.