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Gapdh activity assay kit

Manufactured by Abcam
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The GAPDH activity assay kit is a biochemical tool used to measure the enzymatic activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in biological samples. GAPDH is a key enzyme involved in the glycolytic pathway. This kit provides a quantitative colorimetric method to determine GAPDH activity.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Streptomycin, by Thermo Fisher Scientific (3 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Gpx assay kit, by Cayman Chemical (2 mentions) Pdi assay kit, by Enzo Life Sciences (2 mentions) Pyruvate, by Corning (1 mentions) Complete rpmi, by Corning (1 mentions) Adenosine triphosphate (atp), by Merck Group (1 mentions) Lipopolysaccharides (lps), by InvivoGen (1 mentions) Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions) Glutamine, by Corning (1 mentions) Hepes, by Corning (1 mentions) Bca kit, by Thermo Fisher Scientific (1 mentions) Sulforhodamine b, by Merck Group (1 mentions) High glucose dulbecco s modified eagle s medium, by Fujifilm (1 mentions) Heptelidic acid, by Adipogen (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Bradford protein assay, by Bio-Rad (1 mentions) Imark microplate absorbance reader, by Bio-Rad (1 mentions) Lysostaphin, by Merck Group (1 mentions)

24 protocols using gapdh activity assay kit

1

Analyzing Inflammasome Activation in T2DM

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Human peripheral blood mononuclear cells (huPBMCs) from normal control (ctrl) and type 2 diabetes mellitus (T2DM) patients were purchased from Precision for Medicine, inc (BuyPBMC). Cells were maintained in vapor phase of liquid nitrogen (−120°C). On the day of experiment, huPBMCs were thawed in 37°C water bath and gently pipetted into 10mL of complete RPMI (Corning) supplemented with 10% FBS (Atlanta Biologicals), 2mM glutamine (HyClone), 1mM HEPES (Corning) and 1mM pyruvate (Corning) and centrifuged at 500g for 10min. Media was aspirated and cells were resuspended in 1mL of complete RPMI with supplements. 100,000 cells were plated in each well of a 96 well plate and incubated overnight. Stimulation with LPS (Invivogen) was performed the next day. 200ng/ml of LPS was given to the cells for 6hrs with or without 1400W (50μM). Cells were then treated with 2mM ATP (Sigma) for 30min and media was collected for huIL-1β ELISA (DY201–05). Cellular GAPDH Activity was assessed using GAPDH activity assay kit (abcam, ab204732).
De Jesus A., Keyhani-Nejad F., Pusec C.M., Goodman L., Geier J.A., Stoolman J.S., Stanczyk P.J., Nguyen T.A., Xu K., Suresh K.V., Chen Y., Rodriguez A.E., Shapiro J.S., Chang H.C., Chen C., Shah K.P., Ben-Sahra I., Layden B.T., Chandel N.S., Weinberg S.E, & Ardehali H. (2022). Hexokinase 1 cellular localization regulates the metabolic fate of glucose. Molecular cell, 82(7), 1261-1277.e9.
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2

Quantifying GAPDH Activity in Cortical Tissue

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GAPDH activity was quantified using a GAPDH activity assay kit (Abcam, Cambridge, UK). Cortical tissue was homogenised in a GAPDH assay buffer (final concentration: 30 μg tissue/100μL buffer) and enzyme activity was assessed in a 96-well plate according to manufacturer’s instructions. Absorbance was measured at 450 nm in kinetic mode at 37 °C. A total of 21 readings were recorded over 40 min and GAPDH activity was calculated according to the manufacturer’s instructions. Protein quantification of lysates was performed using the standard BCA Kit (Pierce, Thermo Fisher Scientific, Warrington, UK), and data are expressed as U/mg of total protein.
Mela V., Sayd Gaban A., O’Neill E., Bechet S., Walsh A, & Lynch M.A. (2022). The Modulatory Effects of DMF on Microglia in Aged Mice Are Sex-Specific. Cells, 11(4), 729.
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3

Quantifying GAPDH Activity in Salmonella

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GAPDH activity in Salmonella was measured by the GAPDH activity assay kit (Abcam) as per manufacturer’s instructions. Briefly, Salmonella were grown in MOPS-GLC medium at 37°C to an OD600 of 0.25. Cells were sonicated in 200 μl of ice-cold lysis buffer (25 mM Tris-HCl (pH 8.0),100 mM NaCl). Insoluble material was removed by centrifugation at 13,000 g for 10 min at 4°C. GAPDH enzymatic activity in soluble cytoplasmic extracts was estimated by measuring the accumulation of NADH at 450 nm formed in conversion of glyceraldehyde-3-phosphate into 1, 3-bisphosphate glycerate. GAPDH activity was standardized to equal amounts of protein. GAPDH activity was calculated by linear regression using known NADH standard.
Kant S., Till J.K., Liu L., Margolis A., Uppalapati S., Kim J.S, & Vazquez-Torres A. (2023). Gre factors help Salmonella adapt to oxidative stress by improving transcription elongation and fidelity of metabolic genes. PLOS Biology, 21(4), e3002051.
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4

B16F10 Cell Proliferation Assay

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B16F10 cells were obtained from Dr. Takayuki Ohkuri (Department of Pathology, Asahikawa Medical University, Asahikawa, Japan.). High-glucose Dulbecco’s modified Eagle’s medium was purchased from FUJIFILM Wako Pure Chemical, Osaka, Japan. Fetal bovine serum, L-glutamine, penicillin, and streptomycin were purchased from Thermo Fisher Scientific, MA, USA. Sulforhodamine B was purchased from Merck KGaA, Darmstadt, Germany. Heptelidic acid was purchased from Adipogen Life Sciences, CA, USA. A GAPDH activity assay kit was purchased from Abcam, Cambridge, UK.
Isozaki S., Konishi H., Tanaka H., Yamamura C., Moriichi K., Ogawa N, & Fujiya M. (2022). Probiotic-derived heptelidic acid exerts antitumor effects on extraintestinal melanoma through glyceraldehyde-3-phosphate dehydrogenase activity control. BMC Microbiology, 22, 110.
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5

Quantifying GAPDH Enzymatic Activity

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GAPDH enzymatic activity was determined by GAPDH Activity Assay Kit (ab204732, Abcam) following the manufacturer’s instructions. GAPDH catalyzes the conversion of glyceraldehyde-3-phosphate (GAP) to 1, 3-bisphosphate glycerate (BPG) and the intermediate, NADH, which reacts with a developer to form a colored product that absorbs maximally at 450 nm. The activity of GAPDH can be reflected by measuring NADH production at 450 nm.
Jin X., Li X., Li L., Zhong B., Hong Y., Niu J, & Li B. (2022). Glucose-6-phosphate dehydrogenase exerts antistress effects independently of its enzymatic activity. The Journal of Biological Chemistry, 298(12), 102587.
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6

GAPDH Enzymatic Activity Assay

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Recombinant human GAPDH (rhGAPDH) (Abcam, ab 82633) was used. 2 μg of rhGAPDH was transferred to HEN buffer (250 mM Hepes‐NaOH, 1 mM EDTA 0 and 1 mM neocuproine) and incubated with PBS or GP‐2250 (100 and 250 μM) at 37°C. After 0, 30 and 60 min of incubation, an aliquot was taken for the enzyme assay. The assay was conducted using the GAPDH Activity Assay Kit (Abcam Cambridge, UK, ab204732) following the manufacturer's instructions.19
Majchrzak‐Stiller B., Buchholz M., Peters I., Waschestjuk D., Strotmann J., Höhn P., Hahn S., Braumann C., Uhl W., Müller T, & Möhler H. (2023). GP‐2250, a novel anticancer agent, inhibits the energy metabolism, activates AMP‐Kinase and impairs the NF‐kB pathway in pancreatic cancer cells. Journal of Cellular and Molecular Medicine, 27(14), 2082-2092.
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7

GAPDH Activity Assay of CdS QD Exposure

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The activity of GAPDH was determined using a GAPDH Activity Assay Kit (Abcam, Cambridge, UK) following the manufacturer’s instructions. The assay is based on spectrophotometric measurement of NADH formation catalysed by GAPDH. Cultures were grown for 9 and 24 h in the presence and absence of 100 mg L−1 CdS QDs were diluted to the same OD600 value of 1. Forty-five microlitre was used for the assay and the reaction was run for 60 min at 37 °C. The absorbance of the reaction mixture was measured at 450 nm in kinetic mode using the iMark™ Microplate Absorbance Reader (Bio-Rad). GAPDH activity (U) was calculated as the amount of NADH produced (nmol) per unit of time (min) and was normalised to the protein content of the whole-cell lysate determined by the Bradford Protein Assay (Bio-Rad Laboratories Inc., Hercules, CA, USA).
Gallo V., Srivastava V., Bulone V., Zappettini A., Villani M., Marmiroli N, & Marmiroli M. (2020). Proteomic Analysis Identifies Markers of Exposure to Cadmium Sulphide Quantum Dots (CdS QDs). Nanomaterials, 10(6), 1214.
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8

MRSA Oxacillin Exposure and GAPDH Activity

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A single MRSA colony was planktonically cultured in LB (Invitrogen) medium containing oxacillin (6 μg/ml; Sigma‐Aldrich Co.) for 24 h. MRSA (2 × 107 CFU) was lysed by application of Bacterial Protein Extraction Reagent (B‐PER; Thermo Fisher Scientific, Inc. catalog no. 78243) containing lysozyme (5 mg/ml; Thermo Fisher Scientific, Inc. catalog no. 90082), lysostaphin (0.5 mg/ml; Sigma‐Aldrich Co. catalog no. L7386), and Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Inc.) for 37°C for 1 h. Proteins were isolated by centrifugation (20,784 g for 10 min; Thermo Fisher Scientific, Inc.) and treated with DMF (150, 300, and 600 μg/ml) for 1 h. GAPDH activity was subsequently measured using a GAPDH activity assay kit (Abcam, Cambridge, MA, USA, catalog no. ab204732) according to the manufacturer's instructions. Absorbance (450 nm) was detected using BioTek Cytation™ 5 Cell Imaging Multi‐Mode Reader and analyzed using BioTek Gen5 software (BioTek Instruments Inc.).
Kwon H., Yu K.E., Cahill S.V., Alder K.D., Dussik C.M., Kim S., Sharma L., Back J., Oh I, & Lee F.Y. (2022). Concurrent targeting of glycolysis in bacteria and host cell inflammation in septic arthritis. EMBO Molecular Medicine, 14(12), e15284.
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9

GAPDH Activity Assay in B16F10 Cells

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The activity of GAPDH was determined using a GAPDH Activity Assay Kit (Abcam) following the manufacturer’s instructions. B16F10 cells were seeded onto 12-well microplates at 1 × 105 cells/well and cultured for 24 h. After 24 h of HA treatment (n = 3) at different HA concentrations (0.025, 0.05, 0.25, 0.5, and 2.5 μg/mL), the cells were lysed using GAPDH assay buffer and the its activity was determined (mU/mL). The GAPDH activity in tumors from B16F10 graft mice model was also determined following the manufacturer’s protocol.
Isozaki S., Konishi H., Tanaka H., Yamamura C., Moriichi K., Ogawa N, & Fujiya M. (2022). Probiotic-derived heptelidic acid exerts antitumor effects on extraintestinal melanoma through glyceraldehyde-3-phosphate dehydrogenase activity control. BMC Microbiology, 22, 110.
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10

GAPDH Activity Assay Protocol

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The enzyme activity of GAPDH was determined using a GAPDH Activity Assay Kit (Abcam, Cambridge, England) following the manufactures’ protocol. The assay is based on colorimetric measurement of NADH formation catalyzed by GAPDH. One million cells (106) in 10 mL medium were seeded into 35 mm petri-dish and treated with ascorbate. Cells or mouse tumor tissues were suspended in GAPDH assay buffer and subjected to homogenization and sonication. The cytosolic fraction was prepared by centrifuging the lysate at 20,000 g for 15 minutes at 4 °C, and then 20 μL were used for each reaction. The samples were incubated with GAPDH substrates and a color developer provided at 37 °C, and the OD450 nm was measured in kinetic mode for 10 minutes. For the calculation of NADH production, a standard curve was generated using NADH amount ranging from 2.5 to 12.5 nmol/reaction. Two time points in the liner range of the sample curve were used for the calculation. A positive control was provided in the kit. Background controls were samples incubated with the color developer but without GAPDH substrate. GAPDH activity (U) was calculated as the amount of NADH production (nmol) in unit time (minute), and was normalized to the protein content of the whole-cell lysate detected by the bicinchoninic acid protein method (Pierce Biotechnology).
Ma E., Chen P., Wilkins H.M., Wang T., Swerdlow R.H, & Chen Q. (2017). Pharmacologic ascorbate induces neuroblastoma cell death by hydrogen peroxide mediated DNA damage and reduction in cancer cell glycolysis. Free radical biology & medicine, 113, 36-47.
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