Preparation and characterization of polyclonal antibodies against rat sodium pump alpha 2 subunit M1-M2 extra membrane fragment / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To prepare polyclonal antibodies against sodium pump alpha 2 subunit M1-M2 extramembrane fragment (NKAα2 EM1) for studying the pathogenesis of hypertension.</p><p><b>METHODS</b>According to the GenBank data, the amino acid sequence of NKAα2 EM1 was obtained and the target peptide (LAAMEDEPSNDN) was synthesized using a peptide synthesizer with Fmoc method and purified with high-performance liquid chromatography. The synthesized peptide was then coupled to KLH for immunizing New Zealand white rabbits for 4 times to obtain the antiserum. The IgG antibodies against the synthetic peptide, after affinity purification with Protein A, were used for detecting NKAα2 EM1 expression in rat aortic vascular smooth muscle cells by enzyme-linked immunosorbent assay and immunocytochemistry (ICC).</p><p><b>RESULTS</b>The synthesized peptide fragment , which consisted of 13 amino acid residues including one derivatized cysteine residue in the N-terminal (LAAMEDEPSNDN-C), had a theoretical relative molecular mass of 1408.48 D with a measured relative molecular mass of 1407.90 D and a purity exceeding 85.5%. The titer of the antiserum was more than 1512 000, and the purified IgG antibody concentration was 0.965 mg/ml after purification with Protein A. At a 11000 dilution (final concentration of 1 µg/ml), the titer of the purified IgG antibody was more than 1256 000. The purified IgG antibody could be used at 1100 to 1200 dilutions for for immunocytological examination of formalin-fixed cells.</p><p><b>CONCLUSION</b>The anti-NKAα2 EM1 polyclonal antibodies obtained can be used in ELISA and immunocytochemistry for detecting the sodium pump alpha 2 subunit in formalin-fixed tissue or cells to facilitate investigation of the relationship between sodium pump and hypertension.</p>