Archive for the ‘Plasma’ Category - Part 3

Secreted Epididymal Glycoprotein: RESULTS(1)

Immunocytochemical Localization of 2D6-Reactive Antigens in Epididymal Tissue The epithelium lining the efferent ductules, initial segment, and proximal caput epididymidis did not stain with 2D6 McAb/FITC-RAM (Fig. 1A). However, a strong signal was detected in the microvilli and apical cytoplasm of principal cells in the distal caput epididymidis and thereafter in the epithelium throughout the […]

Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(11)

For internal peptide sequencing, gel slices containing the 24-kDa form of 2D6 antigen were lightly stained with 15-20% Gelcode and trypsinized in situ, and released peptides were analyzed by tandem mass spectroscopy (MALDI-TOF MS/MS) (M-Scan Ltd, Wokingham, UK). Two peptides were successfully sequenced, and the analysis was repeated three times.

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. […]

Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(9)

Analysis of the Epitope Recognized by 2D6 McAb (Protein versus Carbohydrate) The cauda form of 2D6 antigen is highly glycosylated, and since many monoclonal antibodies recognize specific sugar epitopes, a preliminary analysis was carried out to determine whether 2D6 monoclonal antibody was binding to protein or carbohydrate moieties. For this purpose a Western blot containing […]

Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(8)

To investigate the possibility that the form of the 2D6 antigen on cauda spermatozoa had a GPI anchor, washed sperm suspensions (100 |xl) were incubated with 5 U/ml of phosphatidylinositol-specific phospholipase C (PI-PLC) for 2 h at 37°C. Samples were centrifuged and SDS extracts prepared of the supernatant and pellets for SDS-PAGE and Western blotting. […]

Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(7)

Gels were stained with Coomassie blue R250 using standard procedures. Western blots were prepared from parallel gels, blocked with 4% (w/v) nonfat dry milk (‘‘Marvel’’) in PBS for at least 3 h at room temperature, and probed with McAb supernatants or polyclonal antisera (diluted 1:1000) for 2 h. After washing three times in PBS, blots […]

Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(6)

Lymphocytes were prepared from two rat spleens minced in 10 ml aMEM (Gibco, Paisley, uK) containing 10% (v/v) fetal calf serum (FCS; Gibco, UK). Tissue pieces were disaggregated in a Potter homogenizer (four to five strokes). The cell suspension was centrifuged at 1000 X g for 3 min and the supernatant removed and centrifuged at […]

Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(5)

Quantitation of 2D6 McAb Binding to Spermatozoa by Fluorescence-Activated Cell Sorting (FACS) The amount of 2D6 McAb bound to spermatozoa was quantified by FACS analysis using a Beckton Dickinson 2 laser FACScalibur, and the resulting data were analyzed on Cell Quest software (Beckton Dickinson, Franklin Lakes, NJ). Spermatozoa from the specified epididymal region were washed […]

Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(4)

The suspension was gently agitated for 5 min, and the released spermatozoa + fluid was decanted away from tissue fragments and centrifuged at 1000 X g for 5 min. The resulting supernatant was clarified by recentrifugation at 10 000 X g for 5 min and the sperm-free caput or cauda plasma stored frozen at —20°C. […]

Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(3)

Frozen sections were cut at —20°C using a Reichert FC4 cryo-ultramicrotome and incubated in undiluted 2D6 McAb supernatant for 3-5 h at 4°C. Control sections were incubated in culture medium only. After thorough washing in PBS/0.1% (w/v) BSA, sections were incubated in rabbit anti-mouse IgG diluted 1:1000 for 30 min followed by colloidal gold (5 […]

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