TITLE:
Immunobiochemical Characteristics of Purified Native Leptin Protein from Indian Major Carp, Rohu (Labeo rohita Ham.)
AUTHORS:
Seikh Sahanawaz Alam, Siddhartha Narayan Joardar, Ashis Kumar Panigrahi, Thangapalam Jawahar Abraham, Sabyasachi Mukherjee, Anupama Mukherjee
KEYWORDS:
Affinity Chromatography, ELISA, Leptin, MALDI-TOF, Rohu, Western Blot
JOURNAL NAME:
Open Journal of Immunology,
Vol.4 No.4,
November
17,
2014
ABSTRACT: Information regarding molecular characteristics of leptin protein in different animal species in-cluding fish is scarce. With the aim of characterizing the native leptin protein of Indian major carprohu (Labeo rohita), at molecular level, the present study was designed to isolate rohu leptin from its hepatocytes (the prime source of leptin in fish) and immunobiochemical characterization of the same, subsequently. In the present study, chemical treatment and ultra-sonication technique was used for isolating leptin from rohu liver tissue. Purification of the protein was attempted using affinity column chromatography. The molecular, biophysical and serological characterization of rohu leptin was carried out by 2D-gel electrophoreis, SDS-PAGE, MALDI-TOF Mass spectroscopy and Western blot. The SDS-PAGE and 2D gel analysis revealed that rohu native leptin possesses molecular mass of 16 kDa. Western blot analysis showed that the fish hepatocytes possessed the sero-reactive leptin protein of 16 kDa. MALDI-TOF mass spectroscopy and peptide analysis showed the molecular mass of rohu leptin as 16283.38 Da. The serodiagnostic potential of native leptin of rohu was revealed for the first time while assessing its serological responses by ELISA using anti-leptin antibodies.