Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(10)
Purification and Peptide Sequence Analysis of 2D6-Reactive Antigen from Cauda Spermatozoa
Washed caudal sperm were resuspended in PBS/1 mM PefaBloc containing 1 M KCl and incubated at room temperature for 20 min to release non-covalently-bound proteins from the plasma membrane. Spermatozoa were washed twice more in PBS and extracted with 1% NP-40 as described previously. Proteins were separated by SDS-PAGE in the first dimension under nonreducing conditions and in the second dimension under reducing conditions. Gels were either stained directly with Gelcode Blue Reagent (Pierce Chemical Co., Rockford, IL) or Western blotted and stained with Coomassie blue.
Protein bands at ~24 kDa were outlined with pencil and blots destained and probed with 2D6 McAb using 4-chloronaphthol as dye reagent. This procedure enabled the reduced 24-kDa form of 2D6 antigen to be located on the blot with high precision. Subsequently, its position on Coomassie blue-stained gels and blots could be ascertained with accuracy. further
Fifteen pieces of PVDF membrane containing the 24-kDaform of2D6 antigen were pooled and subjected to N-terminal sequence analysis by automatic Edman degradation on an Applied Biosystems 470A gas-phase sequenator equipped with a 120A on-line phenylthiohydantoin analyzer.