Vol. 11, Special Issue 3 (2022)

Author(s):
Manasa Varra, Girish Kumar V, Ramesh HS, Suchitra BR, Sudha G and Pooja CH

Abstract:
Optimization of the methods for urine preparation and precipitation is an essential step in the identification of protein biomarkers pertaining to the physiological state. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis is a proteomic technique which separates proteins based on the molecular weight. Studies have reported that urine precipitation methods can have an influence on the protein recovery. Differences in protein preparation is also found to result in differences in the proteins identified at specific physiological state. In this study, urinary proteins from buffaloes collected at the estrus phase and at diestrus phase were precipitated from smaller volumes using ammonium sulphate, ethanol, acetone and methanol/chloroform followed protein separation by 1D SDS-PAGE. Among the four precipitation methods, ammonium sulphate precipitation is found to be an easy method for the separation of proteins as detected by Coomassie blue staining. With respect to the proteins separated from urine at estrus phase in comparison to the diestrus phase, distinct protein bands were obtained at around 150KDa with ammonium sulphate, ethanol and acetone precipitation indicating the influence of protein preparation and physiological state of sample on the detection of specific proteins separated by SDS-PAGE.