Pavalko FM et al. (1983) Worm Breeder's Gazette "technical note."

Roberts TM, Pavalko FM

[Worm Breeder's Gazette, 1983]

Use of Colloidal Gold Particles in EM We have been using a simple and inexpensive procedure to produce colloidal gold particles of a uniform size (5 nm) which can be conjugated directly to proteins. Gold particles serve as excellent electron dense markers for use in electron microscopy. Preparation of Colloidal Gold Particles by Ultrasonics 1. Dilute 0.1 ml of 1% gold chloride [ H(AuCl4) ] in 50 ml dH20. 2. Make solution neutral with 0.2M K2CO3. 3. Immediately before sonication, add 0.5 ml of 100% ethanol as a catalyst. 4. Carry out sonication at 20 Kc and 125 W by immersing a flat-end probe approximately 1 cm under the surface of the gold solution. 5. After about 2-5 min, the solution attains a pinkish color with maximum intensity at A = 520 nm. Note: To prevent the solution from heating up during sonication, keep it cool in an ice water bath. This helps prevent the formation of larger gold particles. Store at 4 C. Preparation of Gold Labelled Protein 1. Mix 1mg of purified protein with 5ml of colloidal gold solution and incubate for 15 min at room temperature with mixing. 2. Add 10mg BSA, mix, and incubate for another 5 min to prevent aggregation of gold-protein complexes. 3. Add NaCl (100g/l) to make a final concentration of 10g/l. 4. Centrifuge at 1700 g for 20 min to remove excess BSA. 5. Centrifuge supernatant from step 4 at 60,000 g for 15 min to bring down gold labelled protein, leaving unlabelled protein in the supernatant. 6. Pellet should be a loosely packed, dense red sediment and can be resuspended in 0.5 to 1.0 ml of desired buffer. Use undiluted.

Pavalko FM et al. (1983) Worm Breeder's Gazette "amonoclonal and polyclonal antibodies that bind to sperm antigens."

Roberts TM, Pavalko FM

[Worm Breeder's Gazette, 1983]

We have been applying hybridoma technology to our research by generating monoclonal antibodies (MAb's) against C. elegans sperm. We have produced 10 mAb's in our lab and have obtained 2 others from Susie Strome at Boulder. Seven of these mAb's target antigens present on the cell surface. We have been purifying monoclonal antibodies from ascites fluid by DEAE Affi-Gel blue chromatography (supplied by Bio-Rad). Although yields have been lower than ideal (40-60%), these antibodies are free of all contaminants except mouse transferrin. Membrane lipid flow and directed movement of pseudopod surface components on sperm was previously reported by Roberts and Ward (J. Cell Bio. 92: 113-120). We have evidence that the movement of antigen-antibody complexes may be due to a continual flow of membrane lipid (see Bretscher, 1976, Nature 260: 21-23) from the tip of the pseudopod back toward the cell body. Bretscher's lipid flow model of membrane movement predicts that membrane components which diffuse slowly are displaced by the lipid stream whereas those which diffuse rapidly effectively escape lipid flow. Because the rate of diffusion is related to molecular weight, large complexes (e.g. antibody- crosslinked membrane proteins) would be swept along more rapidly than individual molecules (e.g.- unbound membrane proteins). We have tested this prediction by comparing clearance rates of various sized complexes form the cell pseudopod. On sperm, relatively small antigen- mAb complexes clear from the pseudopod surface slowly (2-3 min) while larger, cross-linked antigen-mAb-2 antibody complexes are cleared more rapidly (30-45 seconds). Using mAb's conjugated directly to colloidal gold particles (CGP), we have obtained evidence that new surface antigens are preferentially inserted at the tips of pseudopodial projections and then move rearward toward the cell body pseudopod junction (Pavalko and Roberts, C. elegans Meeting Abstracts, 1983). This observation predicts that there must be a pool of antigen in the cytoplasm which would be available for insertion into the membrane. Labelling thin sections of spermatozoa with CGP conjugates of mAb's 11, 63, and 56 (each of which bind to the cell surface in indirect immunofluorescence assays) reveals antigen on the surface (plasma membrane, exposed face of fused membranous organelles and MO contents). In addition, these antibodies label the cytoplasmic laminar membranes and the pseudopod cytoplasm. Many of the gold particles in the cytoplasm lie just beneath the plasma membrane suggesting that they are bound to antigens that were destined for insertion onto the surface. Because there are no vesicles in the cytoplasm, the mechanism which transports these antigens is unknown. We will be trying to characterize the antigens targeted by the mAb's and to solve the question of how they are shuttled around the cell.

Ward S et al. (1983) Worm Breeder's Gazette "Monoclonal and Polyclonal Antibodies that Bind to Sperm Antigens"

Hogan E, Ward S

[Worm Breeder's Gazette, 1983]

We have examined a number of monoclonal and polyclonal antisera generously provided to us by other labs in order to assess their binding to sperm antigens. The antisera include the following: two rabbit sera and eight mouse monoclonals raised against muscle proteins by Ross Francis: five anti-sperm mouse monoclonals prepared by Susi Strome and obtained initially from Michael Klass; eight anti-sperm mouse monoclonals prepared by Fred Pavalko and Tom Roberts; and a rabbit anti-glyceraldehyde phosphate dehydrogenase (GAPDH) serum obtained from Patrice Yarborough and Ralph Hecht. Our interest in these sera is to use them to identify new sperm antigens whose cellular localization and developmental history can be compared with the sperm specific antigens we have alrealy examined. All the antisera were first assayed for tissue specificity by a solid phase immunoassay with proteins from sperm, eggs, larvae or fem- 1( hc17) hemaphrodites which have no sperm. Those antisera that showed significant binding to sperm proteins were then characterized further by immunostaining of sperm or fem-1 proteins transferred from 1-D or 2- D gels to nitrocellulose ('Western transfers'). In addition the cellular localization of antigens was determined by immunofluorescence.