Baker et al., Anal. Biochem 190:360 (1990)
Carlos Aréchiga, Luciano Bonilla and P.J. Hansen
Dept. of Animal Sciences, University of Florida
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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 dissolved in DMSO. Store at -20oC in 5 ml aliquots for 3 months.
3. Glutathione reductase. Dissolve in buffer to 200 U/ml and store @ 4oC.
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|
|3.125||100 microliters of 6.25 stock||100|
|1.5625||50 microliters of 6.25 stock||100|
|0.625||100 microliters of 6.25 stock||900|
|0.250||10 microliters of 25 stock||990|
|0.150||600 microliters of 0.25 stock||400 microliters of water|
4. Reaction mixture: Mix 1 ml of DNTB, 1 ml of NADPH, 1.15 ml of buffer and 0.02 ml of GSH reductase (good for 31 tubes - make larger amounts if needed).
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.
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 until the blastocyst stage). We try to collect these in as small a volume as possible using a wiretrol and then bring to 5 Ál with water and store at -20 C. Sometimes, we need to collect embryos from more than one drop and then final volume may have been greater than 5 Ál.
3) We do the assay in a microtiter
plate reader that has a repeated-reads function (i.e., we can measure absorbance
at standard time intervals). We usually pick the time point giving best
standard curve retrospectively from each assay. If you want to read absorbance
at only one time, you will need to play around with optimal incubation
© C.F. Arechiga, L. Bonilla, and P.J. Hansen