Meeting Abstract
Petrolisthes cinctipes and P. manimaculis are two closely related species of anomurans that live in the upper intertidal zone along the Central California coast. The objective of this study is to isolate, purify, and characterize vitellins from these crabs, characterize anti-vitellin, and develop an ELISA. Vitellin, an egg yolk protein, is metabolized from a larger hemolymph protein, vitellogenin (Vg). We set to describe the egg yolk proteins of these two animals. Vitellin was isolated from homogenized ovaries through a series of centrifugations and salting out extraneous proteins with saturated ammonium sulfate. After dialysis, SDS-PAGE was used to determine vitellin subunit composition. Interestingly, the conspecifics, P. cinctipes and P. manimaculis, vitellin consist of three major subunits that have a MW of 91±2 kDa, 82±2 kDa, and 65.7±1.4 kDa. Two minor bands were also detected at 111±2.3 kDa and 40±1.3 kDa. It is possible that these minor bands are either a dimer of the three main bands or a metabolite of a larger band. Through gel filtration chromatography, the native molecular mass of P. cinctipes vitellin was found to be 301±14 kDa with a small doublet. The native molecular mass for P. manimaculis vitellin is 324±11 kDa with a more pronounced doublet of 160±13 kDa. A Western blot was used to test the reactivity of the Petrolisthes vitellin with various antibodies. It was found that two of the major Petrolisthes vitellin subunits, 93±2 kDa and 65.7±1.4 kDa, successfully bind with Homarus anti-vitellin antibodies. It is now possible to develop an ELISA that can be used to measure the concentration of vitellogenin in the hemolymph of both species.
