ON TESTIS AND EPlDlDYMlS OF RATS - Pondicherry University ...

3.15.4 Determination of protein
Protein contents were determined according to the method of Lowry el ul.
(1951). The pellet obtained after the extraction of nucieic acid was dissolved in 10
mL of 0.1 N sodium hydroxide solution (see Appendix 1.12). An aliquot of 0.1 mL of
the extract was taken and was made up to I mL with distilled water. Alkaline copper
reagent, 5 rnL, was added to the tubes containing protein extract and then vortexed,
they were allowed to stand for 10 min at room temperature.
Folin-Ciocalteau reagent IN was added to the above samples, vortexed and
allowed to stand for 20 min in dark. The optical density was read at 610 nm in a
Systronics Spectrophotometer. A standard calibration cwe was prepared using
different concentrations of bovine serum albumin.
3.16 Subcellular fractionation of testis
Mitochondrial and microsome-rich fractions of testis were obtained by the
method of differential centrifugation as described by Chainy e/ a/. (1997). Briefly, a
20% (w/ V) homogenate was prepared in ice-cold 0.25 M sucrose solution (see
Appendix 1.13) with the help of a motor-driven glass teflon homogenizer (Potter
Elvehjem type, Rerni). In order to obtain nuclear pellet the homogenate was
centrifuged at 1000 g for 10 min at 4°C.
Mitochondrinl pellets was oblaincd by ccntril'uging post-nuclcar supcrn:it;lnt at
10.000 g for 10 min at 4'C. Microsome-rich fraction was prepared by calcium
chloride (CaCIz) sedimentation method (Kamath and Narayan. 1972). Postmitochondria1
supernatant was diluted with ice-cold CaC12 (I M) so that the final
concentration of CaC12 was 0.8 M. Incubated at 4°C was done for 10 min with
occasional stirring. The sample was then centrifuged at 10,000 g for 10 rnin at 4°C
and the microsome-rich pellet was obtained and dissolved in 0.25 M sucrose solution
(I mg protein1 0. l mL).