Laboratory of P.J. Hansen,Dept. of Animal Sciences, University of Florida
http://www.animal.ufl.edu/hansen/protocols/gsh.htm
The following assay is modified from that developed by Baker et al. (Anal. Biochem. 190, 360, 1990). See that paper for more details of the principle of the method.
Materials (from Sigma)
NADPH, pre-weighed vials (N 7505)
DTNB (D 8130)
GSH reductase, from bakers yeast (G 3664)
GSH, free acid (G 6529)
Prepare in Advance
1. 100 mM NaPO4 buffer, pH 7.5 w/ 1 mM EDTA. Store @ room temperature.
2. 1 mM DNTB. Store frozen in 5 ml aliquots.
3. Glutathione reductase. Dissolve in buffer to 200 U/ml and store @ 4oC.
Prepare Day of Assay
1. 1 mM NADPH.
2. GSH 4 nmol/ml.
3. GSH standards:
| Final concentration (pmol/well) |
microliters of 4 nmol/ml stock |
microliters of water |
| 200 |
use stock undiluted |
|
| 100 |
100 |
100 |
| 50 |
50 |
150 |
| 25 |
25 |
175 |
| 12.5 |
12.5 |
187.5 |
| 6.75 |
12.5 |
387.5 |
| 3.375 |
100 microliters of 6.75 stock |
100 |
| 1.6875 |
50 microliters of 6.75 stock |
100 |
4. Reaction mixture: Mix 5 ml of DNTB, 5 ml of NADPH, 5.75 ml of buffer and 0.1 ml of GSH reductase.
Assay
1. Use 96-well plates. Pipette 50 µl sample or standard into each well (don't forget a blank w/ 50 µl water).
2. Add 100 ul reaction mixture.
3. Place plate in microtiter plate reader immediately. Set to read @ 405 nm, repeat read @ 2 minute intervals.
Use with Embryos
1. Store embryos in minimal volume (~5 µl) in water at -20 C.
2. For assay, add 5 µl (or same volume as embryos are in) 1.25 M phosphoric acid to well of 96-well microtiter plate. Add embryos to this well and then add 40 µl water and 100 µl reaction mixture.
3. Use standards down to ~0.15 pmol.
4. Use blank with proper amt of phosphoric acid.
Notes on Use of The Protocol
1) We have used the procedure for RBCs and embryos. The procedure as attached uses standards for the RBC assay. For embryos, you need more standards < 1 pmol and less of the higher standards.
2) We try to collect 10 or more embryos per assay determination. The number of embryos needed to get detectable GSH varies with stage of development (GSH decreases as development proceeds). We try to collect these in as small a volume as possible using a wiretrol and then bring to 5 µl with water and store at -20 C. Then for the assay, we mix the embryos with 5 µl H3PO4. Sometimes, we need to collect embryos from more than one drop and then final volume may have been greater than 5 µl. We record the volume and add the same volume phosphoric acid.
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