Pyruvate Kinase Assay Kit

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PubMed

Functional Studies - Pyruvate Kinase Assay Kit (AB83432)

PC-3 and MCF-7 cells were treated with OA at the indicated doses and PK activity was determined 12 hr after OA treatment using ab83432. The average values of three independent experiments were shown as Mean ± SD. *, P<0.05, **, P<0.01.

Image from Liu J et al., PLoS One. 2014;9(3):e91606. Fig S2(A).; doi: 10.1371/journal.pone.0091606. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• Biochemical assay

Lab

Biochemical assay - Pyruvate Kinase Assay Kit (AB83432)

Diagram showing the principles of the Pyruvate Kinase assay method.

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Supplier Data

Functional Studies - Pyruvate Kinase Assay Kit (AB83432)

Pyruvate standard curve using ab83432.

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Supplier Data

Functional Studies - Pyruvate Kinase Assay Kit (AB83432)

Pyruvate kinase samples time line using ab83432.

• FuncS

Lab

Functional Studies - Pyruvate Kinase Assay Kit (AB83432)

Pyruvate measured in mouse tissue lysates after 2 min, 10 min and 20 min incubation time showing quantity (nmol) per mg of tested sample.

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Lab

Functional Studies - Pyruvate Kinase Assay Kit (AB83432)

Pyruvate measured in biological fluid after 2 min, 10 min and 20 min incubation time showing quantity (nmol) per ml of tested sample.

• FuncS

Lab

Functional Studies - Pyruvate Kinase Assay Kit (AB83432)

Colorimetric standard curve : mean of duplicates (+/- SD) with background reads subtracted.

• FuncS

Lab

Functional Studies - Pyruvate Kinase Assay Kit (AB83432)

Pyruvate measured in cell lysates after 2 min, 10 min and 20 min incubation time showing quantity (nmol) per 1 mln of tested cells.

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Lab

Functional Studies - Pyruvate Kinase Assay Kit (AB83432)

Fluorometric standard curve : mean of duplicates (+/- SD) with background reads subtracted.

• Enzyme activity assay

PubMed

Enzyme activity assay - Pyruvate Kinase Assay Kit (AB83432)

Pyruvate Kinase Assay Kit ab83432 used with mouse kidney.

Kuo et al. used Pyruvate Kinase Assay Kit ab83432 to investigate the effect of acrolein on PKM2 modification and PK activity in kidney tissues of HFD-STZ-induced DN mice.

Analysis of PK activity in mouse kidney samples from HFD-STZ-induced DN mice (n = 5) and control mice (n = 5).

Pyruvate kinase activity was assessed using a Pyruvate Kinase Assay Kit (ab83432) (Abcam, Waltham, MA, United States) according to the manufacturer’s instructions. Kidney lysates (50 μg), cell lysates collected from acrolein-treated HK2 cells (50 μg), or 5 μL of acrolein-treated pyruvate kinase (Roche, Basel, Switzerland; equivalent to 0.5 mU) were each used for the analysis of PK activity. The relative PK activity was estimated using a pyruvate standard curve and displayed in mU/mL in relation to a control.

• Enzyme activity assay

PubMed

Enzyme activity assay - Pyruvate Kinase Assay Kit (AB83432)

Pyruvate Kinase Assay Kit ab83432 used with cell culture lysates, and Lactate Assay Kit ab65331 used with cell culture medium.

Li et al. used Pyruvate Kinase Assay Kit ab83432 and Lactate Assay Kit ab65331 with cultured human primary airway smooth muscle cells (ASMCs) derived from healthy donors (#CC-2576, N = 4, passage 2) and Chronic obstructive pulmonary disease (COPD) patients.

PKM2 activity is essential for metabolic reprogramming towards aerobic glycolysis in COPD-derived ASMCs. (A) Pyruvate kinase activity was measured in normal ASMCs with si-RNA mediated HHIP and/or PKM2 knockdown with normalization to protein concentration. (C) Lactate levels in the culture medium were measured in COPD-derived ASMCs transfected with si-Ctrl or si-PKM2.

Pyruvate Kinase Assay Kit (#ab83432) was purchased from Abcam.

Measurement of secreted lactate levels in cell culture media : Cells were seeded into a 96-well plate at the density of 3 × 103 cells/well. The culture medium was then collected 24 h after the seeding of cells. The lactate levels in the medium were measured by the Lactate Colorimetric Assay Kit II (#K627-100, BioVision, Milpitas, CA) [Biovision is an Abcam company, K627 is now sold as Abcam ab65331], and lactate levels were then normalized to DNA content measured in each well at the time of collection.

• Enzyme activity assay

PubMed

Enzyme activity assay - Pyruvate Kinase Assay Kit (AB83432)

Pyruvate Kinase Assay Kit ab83432 used with human prostate cancer cell line LNCaP.

Jiang et al. used Pyruvate Kinase Assay Kit ab83432 to investigate the activity of 2 pyrvuate kinase inhibitor compounds.

The activity of pyruvate kinase in LNCaP cells was inhibited by compound 3h and compound 3K depending on concentration. The values represent the mean ± SD. * p < 0.05, ** p < 0.01, and **** p < 0.0001.

A PK assay kit (ab83432, Abcam, Cambridge, UK) was used to detect PK assay activity in LNCaP cells. In short, according to the guide, protein was extracted from cells (50 x 104), pellets with four volumes of assay buffer and protein were added to a 96-well plate, then the PK assay buffer was added for a volume of 50 μL/well. The prepared reaction was mixed with the assay buffer, substrate mix, enzyme mix, and OxiRed™ probe provided in the PK assay kit, and the 50 μL/well reaction mix was added to the 96-well plate containing the protein. The standard linear curve was determined by serial dilutions of 1 nmol/mL standard pyruvate solution. After the reaction, the absorbance of all standards and samples was determined at a 570 nm wavelength using a VERSA Max Microplate Reader (Molecular Devices Corp., Sunnyvale, CA, USA).

• Enzyme activity assay

PubMed

Enzyme activity assay - Pyruvate Kinase Assay Kit (AB83432)

Alt-Aissa et al. used Pyruvate Kinase Assay Kit ab83432 and LDH Assay Kit ab102526 to investigate changes in glycolytic flux in human hearts in coronary heart disease.

Glycolytic pathway in human hearts. (A) Pyruvate Kinase Activity and (B) Lactate Dehydrogenase Activity were measured in lysates of left ventricles of human hearts; Values are expressed as mean +/- SEM expressed as fold change relative to Non-CAD heart lysates; n = 8 in each group; *p < 0.05 vs Non-CAD hearts.

Pyruvate kinase activity was measured in heart lysates from CAD and non-CAD patients. The experiments were performed using the Pyruvate Kinase Assay Kit (ab83432, Abcam, Cambridge, MA, USA) according to the manufacturer instructions.

Lactate Dehydrogenase activity (LDH) was measured in heart lysates from CAD and non-CAD patients. The experiments were performed using the LDH Kit (ab102526, Abcam, Cambridge, MA, USA) according to the manufacturer instructions.

• Enzyme activity assay

PubMed

Enzyme activity assay - Pyruvate Kinase Assay Kit (AB83432)

Pyruvate Kinase Assay Kit ab83432 used with human myoblast cell cultures.

Kuo et al. used Pyruvate Kinase Assay Kit ab83432 to investigate the effect of NANOG over-expression on senescence in human myoblasts.

Pyruvate kinase activity. YM (Young myoblasts), SM (senescent myoblasts cultured for >10 passages), SMN (SM cells over-expressing NANOG) (***p = 0.0005 and ****p < 0.0001). YM : green bars or green lines, SM : red bars or red lines, and SMN : blue bars or blue lines. n = 3 human subjects/group. Data in bar graphs are presented as mean ± SD and data in ECAR plot is presented as mean ± SEM.

Myoblasts were seeded at a density of 5000 cells/cm2 on Matrigel-coated 96-well white plates. The cells were allowed to attach overnight and the next day the cells were starved in a medium containing only 12.5 mM glucose for 6 h. Next, the cells were lysed with Pyruvate kinase assay buffer and PK activity was determined using a PK activity assay kit (ab83432, Abcam, Cambridge, MA) according to the manufacturer’s instructions. The optical density (OD570nm) was measured as a function of time using a BioTek Synergy 4 microplate plate reader at room temperature every 10 min for a total of 40 min.

• Schematic Diagram

Supplier Data

Schematic Diagram - Pyruvate Kinase Assay Kit (AB83432)

Representative image of Pyruvate Kinase Assay Kit ab83432

Components shown from left to right :

- PK Positive Control

- OxiRed Probe

- PK Substrate Mix

- Pyruvate Standard

- Developer Mix A

- PK Assay Buffer

Note : The vial labels shown in this image use generic names for illustrative purposes only and may not exactly match the specific component names included in the kit.

Note : Colors of solutions in image may not precisely match the shade of colors in the actual kit.