Gapdh activity assay kit

Manufactured by Merck Group

The GAPDH Activity Assay Kit is a laboratory equipment product designed to measure the activity of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in biological samples. The kit provides the necessary reagents and protocols to quantify GAPDH activity colorimetrically.

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5 protocols using gapdh activity assay kit

GAPDH Activity Assay Protocol

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The GAPDH activity assay was carried out according to the manufacturer’s protocol (GAPDH Activity Assay Kit, Sigma-Aldrich, cat. no. MAK277). Briefly, a sample containing GAPDH was added to the well of a MaxiSorp 96-well plate (Nunc) in GAPDH Assay Buffer, to which Master Reaction Mix containing GAPDH Developer and GAPDH Substrate in GAPDH Assay Buffer was added. The reaction was carried out for 30 min at 37 °C, measuring absorbance at 450 nm every 1 min. GAPDH activity was recalculated based on the NADH standard curve, where one unit (U) corresponded to the amount of GAPDH enzyme that generated 1 µmol of NADH per minute at pH 7.2 at 37 °C.

Bednarek A., Satala D., Zawrotniak M., Nobbs A.H., Rapala-Kozik M, & Kozik A. (2024). Glyceraldehyde 3-Phosphate Dehydrogenase on the Surface of Candida albicans and Nakaseomyces glabratus Cells—A Moonlighting Protein That Binds Human Vitronectin and Plasminogen and Can Adsorb to Pathogenic Fungal Cells via Major Adhesins Als3 and Epa6. International Journal of Molecular Sciences, 25(2), 1013.

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Glycated GAPDH Deglycation by YajL

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Bacterial GAPDH was incubated overnight at room temperature in a buffer containing 100 mM NaH₂PO₄ pH 7.0, in the presence of 5 mM of MGO. Glycated GAPDH (200 nM) was then incubated with either 100 nM YajL, YajL-GlcNAc, or no enzyme. GAPDH activity was monitored using the GAPDH Activity Assay Kit (# MAK277, Sigma).

El Qaidi S., Scott N.E, & Hardwidge P.R. (2021). Arginine glycosylation enhances methylglyoxal detoxification. Scientific Reports, 11, 3834.

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Glycolysis Enzyme Activities in A375 Cells

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A375 cells were treated with 10 ng/ml IFNγ for 36 hours. Cells were collected and the catalytic activity of glycolysis rate-limiting enzymes were quantitated by Hexokinase Colorimetric Assay Kit (MAK091, Sigma-Aldrich), Phosphofructokinase (PFK) Activity Colorimetric Assay Kit (MAK093, Sigma-Aldrich), GAPDH Activity Assay Kit (MAK277, Sigma-Aldrich), Pyruvate Kinase Activity Assay Kit (MAK072, Sigma-Aldrich), and Lactate Dehydrogenase Activity Assay Kit (MAK066, Sigma-Aldrich), respectively, according to the manufacturer’s instructions.

Mo X., Bailin T, & Sadofsky M.J. (2001). A C-Terminal Region of RAG1 Contacts the Coding DNA during V(D)J Recombination. Molecular and Cellular Biology, 21(6), 2038-2047.

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Proteomic Analysis of Bovine Milk Proteins

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α-Lactalbumin (aLA) from bovine milk (Type I, ≥85%), lysozyme from chicken egg white (LYSO, ≥90%), Rose Bengal (RB), Coomassie brilliant blue G, DL-Dithiothreitol (DTT), tris (2-carboxyethyl) phosphine hydrochloride (TCEP), iodoacetamide (IAM), N-ethylmaleimide (NEM), glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle (GAPDH), catalase from bovine liver and GAPDH activity assay kit were purchased from Sigma Aldrich (St Louis, MO). Beta-2 microglobulin (B2M, > 98%) and recombinant human C-reactive protein (CRP) were obtained from Lee Biosolutions (USA, MO). ¹⁸O water (95.7% pure) was purchased from Sercon. Trifluoroacetic acid (TFA), ammonium bicarbonate, acetonitrile and formic acid for mass spectrometry were obtained from VWR. Beta-2 Microglobulin antibody (4H5L5) and GAPDH monoclonal antibody (GA1R) were obtained from Thermo Fisher. Anti-C Reactive Protein antibody (ab32412) was obtained from Abcam. Sequencing grade trypsin and rLys-C (mass spec grade) were purchased from Promega (Denmark). NuPAGE MES SDS running buffer (20×), NuPAGE LDS sample buffer (4×), NuPAGE 4-12% Bis-tris gel were obtained from Thermo Fisher. Sheep anti-mouse IgG HRP-linked whole Ab (NXA931-1 ML) was obtained from VWR. Anti-rabbit IgG HRP-linked antibody was obtained from Cell Signaling Technology. All solvents employed were HPLC grade.

Jiang S., Carroll L., Mariotti M., Hägglund P, & Davies M.J. (2021). Formation of protein cross-links by singlet oxygen-mediated disulfide oxidation. Redox Biology, 41, 101874.

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mPEG-PCL Nanoparticles for Targeting Mitochondrial Metabolism

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All chemicals were purchased from Saan chemical technology (Shanghai) unless otherwise stated and used without further purification. mPEG₅₀₀₀-PCL₃₀₀₀ and mPEG₅₀₀₀-PCL₃₀₀₀-FA were bought from ShangHai ToYongBio Tech. Inc. N-Hydroxy succinimide, DL-PROPARGYLGLYCINE (PAG), L-Cys, Metformin, Type IV collagenase, GAPDH Activity Assay Kit and Triethylamine were purchased from Sigma-Aldrich. 1,3-Diphenylisobenzofuran and p-Nitrobenzoyl chloride were purchased from TCI (Shanghai). Hydrogen Sulfide (H₂S) Colorimetric Assay Kit was purchased from Elabscience Biotechnology. Mito-Tracker Green and MTT were purchased from Beyotime Biotechnology. Agilent Seahorse XF Cell Mito Stress Test Kits and Agilent Seahorse XF Glycolysis Stress Test Kits were purchased from Agilent Technologies Inc. DCFH-DA was purchased from Solarbio (Beijing). HCT116 cells and human emborynic kidney 293 (HEK293) cells were purchased from Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). All cell lines were routinely tested for mycoplasma contamination, and cells were authenticated by Short Tandem Repeat test.

Zhang Y., Fang J., Ye S., Zhao Y., Wang A., Mao Q., Cui C., Feng Y., Li J., Li S., Zhang M, & Shi H. (2022). A hydrogen sulphide-responsive and depleting nanoplatform for cancer photodynamic therapy. Nature Communications, 13, 1685.

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