for GSH, i follow this protocol

Glutathione was estimated as per methods described earlier (Moron et al., 1929). Glutathione is measured by its reaction with DTNB (5,5’dithio 2-nitrobenzoic acid)(Ellman’s reaction) to give a compound that absorbs light at 412nm. 1.8 ml of 0.2M  Na2HPO4  was mixed with 40 µl 10 mM DTNB and 160 µl of cell homogenate. The blank consisted of distilled water instead of cell homogenate. It was allowed to stand for 2 minutes and the absorbance was read at 412 nm in a Systronic UV-VIS Spectrophotometer.

haven't tried GSSG

all the best

The spectrophotometric method for GSH involves oxidation of GSH 5,5'-dithio-bis(2-nitrobenzoic acid to form the yellow derivative 5'-thio-2-nitrobenzoic acid (TNB), measurable at 412 nm. The glutathione disulfide (GSSG) formed can be reverse to GSH by glutathione reductase in the presence of NADPH.

Rahman I1, Kode A, Biswas SK.Nat Protoc. 2006;1(6):3159-65

More and more labs are using GSH:GSSG ratios as an indicator of oxidative stress.  Measuring reduced glutathione, GSH, is fairly easy as at contains a free thiol group.  We use Ellman’s reagent (5,5'-dithiobis-2-nitrobenzoic acid (DNTB)) that reacts with GSH resulting in a product that can be measured at 412 nm.

 GSSG is more difficult as it is present at a much lower concentration than GSH and it needs to be reduced in order to be measured. We add a thiol scavenger to our samples to bind with GSH to make a pyridinium salt.  This prevents GSH from binding to DNTB and from oxidizing to GSSG.

 We split each sample into two tubes; one for GSH and one for GSSG.  The GSH sample can be measured with Ellman’s reagent in the traditional way. The GSSG sample needs to have the thiol scavenger added (preferably at the time of collection).  DNTB is then added followed by glutathione reductase to convert GSSG to GSH.  While the scavenger is still present, the reaction rate for DNTB is much faster.  The sample can then be measured at 412 nm.

This link has additional information on the method as well as protocols for cuvette and microplate assays.  http://www.oxfordbiomed.com/gshgssg-assay-1

More and more labs are using GSH:GSSG ratios as an indicator of oxidative stress.  Measuring reduced glutathione, GSH, is fairly easy as at contains a free thiol group.  We use Ellman’s reagent (5,5'-dithiobis-2-nitrobenzoic acid (DNTB)) that reacts with GSH resulting in a product that can be measured at 412 nm.

 GSSG is more difficult as it is present at a much lower concentration than GSH and it needs to be reduced in order to be measured. We add a thiol scavenger to our samples to bind with GSH to make a pyridinium salt.  This prevents GSH from binding to DNTB and from oxidizing to GSSG.

 We split each sample into two tubes; one for GSH and one for GSSG.  The GSH sample can be measured with Ellman’s reagent in the traditional way. The GSSG sample needs to have the thiol scavenger added (preferably at the time of collection).  DNTB is then added followed by glutathione reductase to convert GSSG to GSH.  While the scavenger is still present, the reaction rate for DNTB is much faster.  The sample can then be measured at 412 nm.

 This link has additional information on the method as well as protocols for cuvette and microplate assays.  http://www.oxfordbiomed.com/gshgssg-assay-1

http://www.oxfordbiomed.com/gshgssg-assay-1

Thank you all for your valuable suggestions. I have gone through the suggestions. Again I have some questions regarding estimation. For the standard curve of GSH, what should be range? For GSSG, what thiol scavanger should I use and at what concentration. What unit of GR should I use during GSSG estiimation? What should be range of GSSG concentration for standard curve? Thank you all.

Pay attention, GSSG measurement is not an easy task even for skilled researchers, the main risk is an overestimation, with consequent low GSH/GSSG ratios.

NEM can be used as thiol blocking agent. I have published the protocol a couple of years ago.

Analysis of GSH and GSSG after derivatization with N-ethylmaleimide. Nat Protoc. 2013 Sep;8(9):1660-9.

http://www.ncbi.nlm.nih.gov/pubmed/23928499

GSH was estimated in tissues by the method of Sedlak

and Lindsay (1968). Briefly, 5% tissue homogenate were

prepared in 20mM EDTA, pH 4.7, and 100 ml of the

homogenate or pure GSH was added to 0.2M Tris-EDTA

buffer (1.0 ml, pH 8.2) and 20mM EDTA, pH 4.7 (0.9 ml)

followed by 20 ml of Ellman’s reagent (10 mmol/l DTNB in

methanol). After 30 min of incubation at room temperature,

samples were centrifuged, and the absorbance was recorded at

412 nm (Khynriam & Prasad, 2003).

what if DTNB, GSH and KH2PO4(all solid), are available to you? I'm trying to create a protocol and I'm having difficulties doing so

Thanks for all your suggestions and protocols for GSH and GSSG, as I am also need of this information...

Balaraju

Hola! aca te dejo una copia en español de la tecnica de medicion de GSH.

Técnica determinación de Glutatión :

Preparacion de muestras de sangre entera

Técnica determinación de Glutatión preparacion de muestras de sangre entera

Lavar la sangre 3 veces c/ PBS y hacer dilucion de 1:10 con buffer de lisis en baño hielo. separar 20 ul x 3 para Lowry

Mezclar partes iguales de lisado con TCA 10 % (0,5 ml + 0,5 ml) en tubos centrífuga . Centrifugar 10´10.000 g 4-12º C Separar 200 ul de Sobrenadante en tubo reacción por triplicado.

Agregar 1 ml Rvo Ellman. Vortear bien y luego de 5´ leer a 412 nm contra blanco de curva.

Reactivos:

· TCA 10 % y 5 %

· Buffer Na2POH4 0,1 M PH:8 (hemólisis usado para CAT)

· Buffer KPO4H2 0,25 M PH:8

· Reactivo Ellman: DTNB 1,5 mM en buffer KPO4H2 (preparar en el momento).0,0119 gr en 20 ml de buffer fosfato de potasio

· Solucion . Madre GSH: 10 mM en agua dest. (preparar en el día,/ o resiste una sola descongelada, cubrir con papel aluminio porque la luz lo oxida)

Solucion estándar GSH: 0,1 mM en TCA 5 %. Diluir en el momento 10 ul de GSH madre + 990 uL TCA 5%

curva: BSA 2 mg/ml Ptos

Exitos!!!

M*

I am also need the protocols for GSH, So I have been searched the same and I got the information. Thanks for all your suggestions. Thanks Research gate.

IS there any method for GSH isolation?