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Purification of native regucalcin from the seminal vesicular fluid: a calcium binding multi-functional protein.

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Title Purification of native regucalcin from the seminal vesicular fluid: a calcium binding multi-functional protein.
 
Creator Harikrishna, P.
A. M. Shende
Reena K. K
Jobin Thomas
S. K. Bhure
 
Subject Affinity chromatography
Buffalo
Purification
Regucalcin
Seminal vesicular fluid
 
Description Not Available
Regucalcin is a multi-functional protein having roles in calcium homeostasis as well as in anti-apoptotic, anti-prolific and anti-oxidative functions. Recently, it has been reported from the male reproductive tract, but its role in male reproduction needs further investigation; for which the native regucalcin of reproductive origin will be more appropriate. The gel exclusion chromatography followed by diethyl aminoethane cellulose chromatography and two-dimentional cellulose acetate membrane electrophoresis used for its purification are time consuming and less specific. Here, the regucalcin gene from buffalo testis has been cloned, expressed and purified in recombinant form, and subsequently used for raising hyper-immune serum. The Western blot of seminal vesicular fluid probed with anti-regucalcin polyclonal and monoclonal antibodies showed the presence of 28 and 34 kDa bands specific to regucalcin. Further, an affinity matrix has been prepared using anti-regucalcin polyclonal antibodies. An immuno-affinity chromatography method has been standardized to isolate regucalcin from seminal vesicular fluid. The initial complexity of the protein mixture in the seminal vesicular fluid has been reduced by a heat coagulation step. The purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band at 68 kDa that has been further confirmed as regucalcin by Liquid chromatography-mass spectrometry/mass spectrometry. The RGN purified from seminal vesicular fluid will be more appropriate for studying its possible role in male reproduction, especially sperm cell capacitation, hyperactivation, acrosome reaction and cryopreservation. The study can be applied in purifying regucalcin from different tissues or species with minor modifications in the methodology.
Not Available
 
Date 2023-05-30T04:12:38Z
2023-05-30T04:12:38Z
2016-08-01
 
Type Research Paper
 
Identifier Not Available
Not Available
http://krishi.icar.gov.in/jspui/handle/123456789/77870
 
Language English
 
Relation Not Available;
 
Publisher Springer