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.
Cloning and DNA Sequence Analysis of the 24-kDa Antigen
To obtain the full cDNA sequence of the 24-kDa antigen recognized by 2D6 McAb, the rat epididymal cDNA library was screened with a 32P-labeled degenerate oligonucleotide probe 5′ CARGGIGARTAYCARA-CIGAYCCIGCIACIGGIAA 3′ (Y = C or T, R = G or A, I = inosine) encoding a peptide sequence derived from the purified 24-kDa antigen. Bacteria containing inserts that hybridized to the probe were grown in LB ampicillin and plasmids isolated using a Qiagen miniprep kit. The plasmids were then restricted using BamH1 and EcoR1 as directed by the manufacturers (Promega, Southampton, UK) and those with the longest inserts selected for further analysis. DNA sequencing was performed using an ABI automatic sequencer. Ventolin asthma treatment