Pyruvate Kinase Activity Colorimetric,Fluorometric Assay Kit

Details for Product No. ABIN411901
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Target Name (Antigen)
Synonyms CG7070, Dmel\\CG7070, PYK, Pyk, PYK3b, PYK3c
Reactivity
Mammalian
(1), (1), (4), (3), (1), (3), (7), (3), (7), (3), (3), (4), (3)
Minimum Detection Limit 0.1 mU
Application
Activity Assay (AcA)
Pubmed 2 references available
Quantity 100 tests
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Catalog No. ABIN411901
484.00 $
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Purpose Sensitive Assay, HTS
Detection Method Fluorometric,Colorimetric
Specificity The Kit provides a simple, direct and automation-ready procedure for measuring pyruvate kinase activity in various biological samples such as blood, tissues, and culture cells, etc. In the assay, PEP and ADP were catalyzed by PK to generate pyruvate and ATP. The generated pyruvate is oxidized by pyruvate oxidase to produce color (at lambda = 570 nm) and fluorescence (at Ex/Em = 535/587 nm). Since the increase in color or fluorescence intensity is proportional to the increase in pyruvate amount, the PK activity can be accurately measured. The kit detects 0.1 mU/mL pyruvate kinase.
Characteristics Pyruvate Kinase Activity Assay Kit: Colorimetric & Fluorometric Assay for Measuring Pyruvate Kinase Activity in various Biological Samples such as Blood, Tissues, Cultured cells etc. within 30 min. Convenient, Accurate & Reliable.
Components PK Assay Buffer
OxiRed™ Probe (in DMSO)
PK Enzyme Mix
PK Substrate Mix
PK Positive Control (18 mU)
Pyruvate Standard (100 nmol/µL)
Alternative Name Pyruvate Kinase
Background Pyruvate kinase (PK, EC 2.7.1.40) is an enzyme involved in glycolysis. It catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP, yielding one molecule of pyruvate and one molecule of ATP. Lack of pyruvate kinase will slow down the process of glycolysis which causes the disease known as pyruvate kinase deficiency.
Research Area Cancer, Cell Cycle, Kinases/Phosphatases, Metabolism, Enzymes
Comment

Category: Pyruvate
SampleType adds: Testing inhibitor panels, Purified, Isolated enzyme

Assay Time 0.5 h
Protocol 1. Standard Curve Preparations: For the colorimetric assay: Dilute the Pyruvate Standard to 1 nM/µL by adding 10 µL of the Standard to 990 µL of Assay Buffer, mix well. For the fluorometric assay: Dilute the Pyruvate Standard to 1 nM/µL as for the colorimetric assay. Then dilute the standard another 10-fold to 0.1 nM/µL by taking 10 µL into 90 µL of Pyruvate Assay Buffer. Mix well. Add 0, 2, 4, 6, 8, 10 µL of the diluted standard into a series of wells. Adjust volume to 50 µL/well with Assay Buffer to generate 0, 2, 4, 6, 8, and 10 nM/well of the Pyruvate Standard for the colorimetric assay, or 0, 0.2, 0.4, 0.6, 0.8, and 1.0 nM/well for the fluorometric assay.
2. Sample and Positive Control Preparations: Serum can be directly added into sample wells. Tissues or cells can be extracted with 4 volume of the Assay Buffer, centrifuge to get clear extract. Add samples directly into 96 well plate, bring volume to 50 µL/well with PK Assay Buffer. We suggest testing several doses of your sample to ensure the readings are within the linear range. For the positive control (optional), add 5 µL positive control solution to wells, adjust volume to 50 µL/well with Assay Buffer.
3. Reaction Mix Preparation: Mix enough reagents for the number of standard and assays to be performed. For each well, prepare a total 50 µL Reaction Mix containing: Pyruvate Kinase Measurement Background Control* Assay Buffer 44 µL 46 µL Substrate Mix 4 µL ----------- Enzyme Mix 4 µL 2 µL OxiRed™ Probe** 4 µL 2 µL *Pyruvate in the sample will generate background. If significant amount of pyruvate is in your sample, the background control should be performed. The background readings are then subtracted from your sample readings. ** For fluorescent assay, dilute the probe 10X to reduce fluorescence background.
4. Add 50 µL of the reaction mix to each well containing the pyruvate standard, samples and controls, mix well.
5. Measure O.D. 570 nm or fluorescence Ex/Em = 535/587 nm at T 1 to read A 1, measure again at T 2 after incubating the reaction at 25 °C for 10-20 min (or incubate longer time if the PK activity is low in sample) to read A 2, protect from light. The signal increase is due to pyruvate generated by PK, deltaA = A 2 - A 1 Note: It is essential to read A 1 and A 2 in the reaction linear range. It will be more accurate if you read the reaction kinetics. Then choose A 1 and A 2 in the reaction linear range.
6. Calculation: Subtract 0 standard readings from the standards. Plot the pyruvate standard curve. Apply the deltaA to the standard curve to get B nmol of pyruvate generated between T 1 and T 2 by PK in the reaction wells. PK calculation: B PK Activity = x Sample Dilution Factor = nM/min/mL = mU/mL (T1-T2) x V Where: B is the pyruvate amount from pyruvate standard curve (in nmol). T 1 is the time of the first reading (A1) (in min). T 2 is the time of the second reading (A2) (in min). V is the sample volume added into the reaction well (in ml). Unit definition: One unit pyruvate Kinase is the amount of enzyme transfer of phosphate group from PEP to ADP, yielding 1.0 µM of pyruvate per minute at 25 °C.
Restrictions For Research Use only
Storage -20 °C
Expiry Date 12 months
Product cited in: Yang, Xia, Ji et al.: "Nuclear PKM2 regulates β-catenin transactivation upon EGFR activation." in: Nature, 2011 (PubMed).

Parnell, Foulks, Nix et al.: "Pharmacological activation of PKM2 slows lung tumor xenograft growth." in: Molecular cancer therapeutics, 2013 (PubMed).

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