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.

Preparation of Antibodies to Sperm-specific Proteins

S. Ward, D.J. Burke

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.