We want to assay Ascorbate Peroxidase from pathogen stressed wheat leaves. samples are collected after 24, 48, 72 hrs after inoculation
Total reaction mixture suggested to be 2ml consists of .05M potassium phosphate buffer pH 7 .2mM EDTA .5mM ascorbic acid .25 mM H2O2 start your reaction by addition of .1ml of your plant extract at 25 C .record the decrease in absorbance at 290nm for one minute use Extinction coefficient of 2.8mM Cm-1,to calculate the amount of ascorbate oxidized.this method is described by Nakono K and Asada K 1981 in Cell physiology 22 page 867-880
Abdelrahim Ahmad Hunaiti sir for this protocol is it necessary to estimate protein in enzyme extract?
Dear Sudhir you take x gram of you plant tissue mix it with 2X of the potassium phosphate buffer.05 M pH 7 homogenized take the extract centrifuge for 15 min at 10000xg then take 0.1 ml of the. Supernatant as source of enzyme.you can performe protein determination on the supernatant by Lowry or any method you used for protein determination good luck
Ascorbate Peroxidase (AsPOX)
- 0.2 M Tris/HCL buffer (pH 7.8)
- 0.25 mM ascorbic acid
- 0.5 mM H2O2
Parveen Yadav can you help me with a short explanation about 290nm. You write oxidation of ascorbic acid. Do you know an explanation or somenthing about the absorbance in this specific wavelenght? Thank you so much.
Respected sir, Sudhir Navathe Abdelrahim Ahmad Hunaiti Parveen Kumar
I am little confused about the quantity of different chemicals. What should be the quantity in ml/ul to be taken from 0.05M phosphate buffer, 0.2mM EDTA, 0.5mM Ascorbic acid and 0.25 mM H2O2 for final 2 ml reaction mixture in the cuvette ? Please suggest.
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How is ascorbate peroxidase assayed?
Ascorbate peroxidase was assayed by recording the decrease in optical density due to ascorbic acid at 290 nm . The 3 ml reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbic acid, 0.1 mM EDTA, 0.1 mM H2O2, enzyme and water. The reaction was run for 5 min at 25 °C. How can I calculate Ascorbate peroxidase activity?www.researchgate.net/post/How_can_I_calculate_Ascorb…
Which is a specific electron donor for ascorbate peroxidases?
Ascorbate peroxidases (APX; EC 1.11.1.11) exhibit a higher affinity for peroxides (mainly H2 O 2) and utilize ascorbate (AsA) as a specific electron donor in order to reduce H 2 O 2 into H 2 O in organelles, including chloroplasts, cytosol, mitochondria, and peroxisomes. Ascorbate Peroxidase - an overview | ScienceDirect Topics www.sciencedirect.com/topics/agricultural-and-biologica…
What is the final assay concentration of peroxide?
Final Assay Concentrations – In a 3.00 ml reaction mix, the final concentrations are 14 mM potassium phosphate, 0.027% (v/v) hydrogen peroxide, 0.5% (w/v) pyrogallol, and 0.45–0.75 unit peroxidase. Note: Run one cuvette at a time as the maximum rate is in the first minute. 1. Pipette the following reagents into suitable cuvettes: 2.Enzymatic Assay of Peroxidase (EC 1.11.1.7) | Sigma-Aldrich
www.sigmaaldrich.com/technical-documents/protocols/bi…
What is the enzymatic assay of peroxidase?
Enzymatic Assay of Peroxidase (EC 1.11.1.7) Description. This procedure is for the determination of Peroxidase enzymatic activity using Pyrogallol as the substrate. Products using this method include, but are not limited to, P1709, P2088, P6140, P6782, P8125, P8170, P8250, P8375, P8415, and P8651.Enzymatic Assay of Peroxidase (EC 1.11.1.7) | Sigma-Aldrich
www.sigmaaldrich.com/technical-documents/protocols/bi…
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