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Glyceraldehyde 3 phosphate dehydrogenase activity assay kit

Manufactured by Abcam
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The Glyceraldehyde 3 Phosphate Dehydrogenase Activity Assay Kit is designed to measure the activity of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzyme. GAPDH is a key enzyme involved in the glycolytic pathway, catalyzing the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. The kit provides reagents and protocols to quantify GAPDH activity colorimetrically.

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Lab products found in correlation

Pyruvate kinase assay kit, by Abcam (1 mentions) Lactate dehydrogenase assay kit, by Abcam (1 mentions) Hexokinase activity assay kit, by Abcam (1 mentions)

5 protocols using glyceraldehyde 3 phosphate dehydrogenase activity assay kit

1

GAPDH Activity Assay Protocol

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GAPDH-mediated reaction was determined using a Glyceraldehyde 3 Phosphate Dehydrogenase Activity Assay Kit (#ab204732, Abcam, Cambridge, UK) according to the manufacturer’s instructions. Briefly, Reaction mix was added to the extracted samples and incubated at 37 °C for 30 min. NADH levels reflecting GAPDH-mediated responses were quantified by measuring absorbance (OD = 450). Results were normalized with protein concentration and measured using SpectraMax i3.
Kudo K., Greer Y.E., Yoshida T., Harrington B.S., Korrapati S., Shibuya Y., Henegar L., Kopp J.B., Fujii T., Lipkowitz S, & Annunziata C.M. (2024). Dual-inhibition of NAMPT and PAK4 induces anti-tumor effects in 3D-spheroids model of platinum-resistant ovarian cancer. Cancer Gene Therapy, 31(5), 721-735.
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2

Quantifying GAPDH Activity in Yeast

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Yeast strains growing in exponential phase were diluted to the same OD (OD660nm=0.2) in 3 biological replicates and 1ml was collected by centrifugation. Cell lysis was adapted for yeast cells: cell pellets were resuspended in 500μl of cold lysis buffer (20 mM Tris pH8, 15mM EDTA pH8, 15mM EGTA pH8 and 0.1% Triton X-100). Glass beads were added in equal volume (500μl) and cells were lysed by vortexing at 4ºC. Enzymatic activity was quantified twice using the colorimetric Glyceraldehyde-3-Phosphate Dehydrogenase Activity Assay Kit (Abcam) as described by the manufacturer.
Ochoa D., Jarnuczak A.F., Viéitez C., Gehre M., Soucheray M., Mateus A., Kleefeldt A.A., Hill A., Garcia-Alonso L., Stein F., Krogan N.J., Savitski M.M., Swaney D.L., Vizcaíno J.A., Noh K.M, & Beltrao P. (2019). The functional landscape of the human phosphoproteome. Nature biotechnology, 38(3), 365-373.
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3

Quantifying GAPDH Activity in Yeast

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Yeast strains growing in exponential phase were diluted to the same OD (OD660nm=0.2) in 3 biological replicates and 1ml was collected by centrifugation. Cell lysis was adapted for yeast cells: cell pellets were resuspended in 500μl of cold lysis buffer (20 mM Tris pH8, 15mM EDTA pH8, 15mM EGTA pH8 and 0.1% Triton X-100). Glass beads were added in equal volume (500μl) and cells were lysed by vortexing at 4ºC. Enzymatic activity was quantified twice using the colorimetric Glyceraldehyde-3-Phosphate Dehydrogenase Activity Assay Kit (Abcam) as described by the manufacturer.
Ochoa D., Jarnuczak A.F., Viéitez C., Gehre M., Soucheray M., Mateus A., Kleefeldt A.A., Hill A., Garcia-Alonso L., Stein F., Krogan N.J., Savitski M.M., Swaney D.L., Vizcaíno J.A., Noh K.M, & Beltrao P. (2019). The functional landscape of the human phosphoproteome. Nature biotechnology, 38(3), 365-373.
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4

Quantifying Cellular NADH Levels

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The levels of NADH in the cell culture supernatants were assessed using the Glyceraldehyde 3 Phosphate Dehydrogenase Activity Assay Kit (Abcam, Cambridge, UK) according to the manufacturer’s protocols. The levels of NADH were normalized against protein concentration. Data were collected and processed using Gen5 software.
Wu J., Gu X., Zhang J., Mi Z., He Z., Dong Y., Ge W., Ghimire K., Rong P., Wang W, & Ma X. (2022). 4-OI Protects MIN6 Cells from Oxidative Stress Injury by Reducing LDHA-Mediated ROS Generation. Biomolecules, 12(9), 1236.
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5

Glycolytic Enzyme Activity Assays

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The Hexokinase Activity Assay Kit (Abcam ab136957), Phosphofructokinase Activity Assay Kit (Abcam ab155898), Glyceraldehyde 3 Phosphate Dehydrogenase Activity Assay Kit (Abcam ab204732), Pyruvate Kinase Assay Kit (Abcam ab83432), and Lactate Dehydrogenase Assay Kit (Abcam ab102526) were used and accompanying protocols were followed. Briefly, cells from each lineage were trypsinized, counted, and 250,000 cells were collected for each assay. Cells were washed with PBS, homogenized, and the supernatant was mixed with substrate and probe developer. Absorbance was read at 450nm, and activity rate was calculated by measuring absorbance at two time points and compared to a standard curve. Negative and positive controls were used.
Mathur D., Liao C., Lin W., La Ferlita A., Alaimo S., Taylor S., Zhong Y., Iacobuzio-Donahue C., Ferro A, & Xavier J.B. (2023). The Ratio of Key Metabolic Transcripts Is a Predictive Biomarker of Breast Cancer Metastasis to the Lung. Cancer Research, 83(20), 3478-3491.
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