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M2VP accomplishes complete scavenging of GSH in less than one minute. All other methods systematically overestimate GSSG concentrations. Before you measure GSSG, be sure you have M2VP.

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Frequently Asked Questions for BIOXYTECH®
cGPx-340 (Cellular Glutathione Peroxidase) Assay
Catalog Number: 21017
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How long does the test take to perform?
Preparation: 15 minutes Assay Procedure: 4 minutes

What is the assay range for the GPx-340 method?
0.035 to 0. 15 A340/minute, this corresponds to approximately 5.6 to 24 mU/mL enzyme activity.

What samples can be tested for Glutathione Peroxidase?
Purified enzymes or clear supernate from cellular lysate or tissue homogenate. For testing GPx in plasma, OXIS supplies the pl

How are samples prepared prior to testing?
General Sample Preparation Protocol. Homogenize the sample in 4-8 volumes (per weight tissue) of cold buffer (PBS). Centrifuge (e.g., 5000-10,000 x g) for 10-20 minutes at 2-8

What is the stability of the assay reaction?
The assay requires kinetic measurement to yield a rate.

What concentrations of GPx can be found in various samples?
Concentrations vary depending on the model system and sample type.

What control is needed to perform the test?
No control is required however if one is desired, OXIS supplies Cellular Glutathione Peroxidase Control, Catalog Number 27617.

What is the shelf life of the GPx-340 kit?
6 months from the date of manufacture when NADPH reagent is stored frozen. All other components are refrigerated.

How is GPx-340 calculated in the procedure?
Determine the rate of decrease in A340 per minute by either: Calculating the difference a A340 ran between 60 and 120 seconds or by averaging the rate of change for several timed intervals (e.g,, 15 second or 30 second intervals), Doing regression analysis on A340 as a function of time to obtain the slope (rate). Calculate the net rate for the sample by subtracting the rate observed for a water blank (water instead of sample). Convert the net A340/min for the sample to NADPH consumed (nmol/min/mL) using the following relationship: 1 mU/mL = 1 nmol NADP/min/mL - (A340/min)/0.00622 Correct for the dilution of the sample: 16 fold dilution in the assay (7OgL to 1120gL). Dilution of sample prior to adding to the cuvette. Note: Total dilution is 16 x sample dilution (prior to assay). Express the units of activity in the original sample per ml, or in relationship to the protein or hemoglobin content (for erythrocytes).

What are the advantages of the GPx-340 method?
Simple, easy to use protocol. With the exception of the control, all reagents necessary to run the assay are provided in the kit.

Can the GPx-340 assay used to detect total GPx protein expression?
The cGPx assay measures activity not total protein. Therefore if the protein has undergone a conformational change etc. it will not be picked up in the assay.

Can the cGPx assay detect plasma glutathione peroxidase?
Yes, even though plasma GPx is different than the cellular form it will react in the cGPx assay. The researcher needs to be aware that there are other substances in plasma such as serum albumins which can interfere causing over estimation of plasma GPx activity. pl

What is the principle of the assay?
The GR-340 assay is based on the oxidation of NADPH to NADP+ catalyzed by a limiting concentration of glutathione reductase. One GR activity is defined as the reduction of one micromole of GSSG per minute at pH 7.6 and 25

How long does the test take to perform?
Preparation: 5 minutes Assay Procedure: 6 minutes

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