Abcam’s Thioredoxin Reductase 1 (TXNRD1) Activity Assay kit is designed for the sensitive and accurate measurement of Thioredoxin Reductase 1 activity in Human cell and tissue extracts.
The assay uses a 96-well plate with an antibody specific to isoform 1 of thioredoxin reductase to isolate the isozyme pre-coated onto the wells. Samples are added to the wells and incubated at room temperature, any Thioredoxin Reductase 1 present in the sample will be immobilized in the well. After washing, the Reaction Buffer is added to each well and enzyme activity is measured. By analyzing the enzyme’s activity in an isolated context, outside of the cell and free from other isoforms, an accurate measurement of the enzyme’s functional state can be understood.
Thioredoxin Reductase 1 (TXNRD1) is a selenium-containing enzyme part of the thioredoxin system responsible for regulating oxidative stress and redox signaling via reduction of disulfide bonds. The dimer, Thioredoxin Reductase 1, reduces thioredoxin and other substrates using NADPH and an FAD cofactor. Thioredoxin Reductase 1 involvement in protecting against oxidative stress and injury, regulation of cellular development and growth, and various other cellular processes make it an interesting target for studies of various cancers, AIDS, and other diseases. Humans express three different isozymes of thioredoxin reductase, isoform 1 is the cytosolic form, isoform 2 is the mitochondrial form, and isoform 3 is a testes specific form.
The enzyme activity is determined by following the NADPH-assisted reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to 5-thio-2-nitrobenzoic acid (TNB) which leads to increased absorbance at 412 nm. Two moles of TNB are formed from the oxidation of one mole of NADPH according to the reaction shown below.
DTNB + NADPH + H+ ↔ 2 TNB + NADP+
The molar extinction coefficient of TNB is 14.15 X 103 mM-1cm-1. Thioredoxin Reductase 1 activity is controlled by enzyme amount. An antibody specific to isoform 1 of thioredoxin reductase is used to isolate the enzyme from a bulk protein source for isoform-specific enzymatic activity measurements. Because the activity measured is specific to the isolated enzyme, a thioredoxin reductase inhibitor is not required for background signal subtraction.
|Components||1 x 96 tests|
|10X Blocking Buffer||1 x 8ml|
|10X Buffer||1 x 25ml|
|200X DTNB||1 x 110µl|
|200X NADPH||1 vial|
|5X Activity Solution||1 x 5ml|
|Extraction Buffer||1 x 15ml|
|TXNRD1 96-well microplate||1 unit|
Our Abpromise guarantee covers the use of ab190804 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Functional Studies||Use at an assay dependent concentration.
Quantitative activity assay