effect of infection of the filarial parasite brugia malayi - Pondicherry ...

Reagent6
1. DTNB : 0.6 mM in 0.2 M phosphate buffer, pH 8.0.
2. Phosphate buffer : 0.2 M, pH 8.0.
3. TCA : 5%
4. Standard : 10 mg of reduced glutathione in I00 ml water. This contained
10pgm of glutathione in 0. I ml solution.
To 0.1 mi of the homogenate, 1.0 ml of water and 2.0 ml of precipitating agent were
added, mixed thoroughly and centrifuged after 5 minutes. To an aliquot of the supernatant,
2.0 ml of DTNB reagent was added and made upto a final volume of 3.0 ml with phosphate.
buffer. The absorbance was read at 412 nm against a reagent blank The amount of
glutathione was exprrssed as pgm of glutathionel mg protein.
4.1.1.8 Total thiol groups
(1 %8).
Total thlol groups were estimated acconhg to the method of Sedlack and Lindsay
The sulphydryl pups in tissues were determined by using the Ellman's reagent. In
this method, DTNB is reduced by -SH groups to form 1 mole of 2-nitro, 5-mercaptobenzoic
acid per mole of -SH.
Reagents
1.0.01 MDTNEI : 99 mg DTNEI dissolved in 25 ml of absolute methanol.
2. Tris-HCl buffer : 0.2 M, pH 8.2 containing 0.02 M EDTA.
The sampk was pnpartd by homogenising 100 mg of tissue in 4.0 ml of 0.02 M
EDTA. To an aliquot of the tissue homogenate, 1.5 ml of Tris-HCI buffer @H 8.2) and 0.1
ml of DTNB was added, mixed and made upto 10.0 ml with absolute methanol. A reagent
blank without the sample and sample blank without DTNEI were prepared in the same
manna. The test tubes were stoppered and allowed to stand with occasional hkmg for 15
minutes. The reaction mixture was cenhifuged at 3000g at R.* for 15 minutes. The