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Phosphofructokinase pfk activity colorimetric assay kit

Manufactured by Merck Group
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The Phosphofructokinase (PFK) Activity Colorimetric Assay Kit is a laboratory test kit used to measure the activity of the enzyme phosphofructokinase. Phosphofructokinase is a key enzyme involved in glycolysis, the metabolic pathway that converts glucose into energy. The kit uses a colorimetric method to quantify the activity of this enzyme in biological samples.

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5 protocols using phosphofructokinase pfk activity colorimetric assay kit

1

Measuring PFK Activity in Streptococcus thermophilus

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PFK activity was measured on the total protein extract of EdCs using the Phosphofructokinase (PFK) Activity Colorimetric assay kit (Sigma-Aldrich, Milan, Italy). To this aim, EdCs were prepared as described previously and subsequently subjected to total protein extraction. Mechanical cell disruption was carried out using a Precellys bead beater (Advanced Biotech Italia SRL, Seveso, Italy). To minimize the interference of NADH-generating enzymatic activities, the PFK assay was performed in the presence of 30 nM Br-Acivicin, a chemical inhibitor of glyceraldehyde-3-phosphate dehydrogenase (43 (link)). Several sample dilutions were tested to ensure that the readings were within the linear range of the standard curve. After this setup, S. thermophilus EdCs total protein extract diluted 1 to 200 in PFK buffer was used in the reaction. When necessary, NH3 or NH4Cl was added to the reaction mix to reach a final reaction concentration of 0.5 mM. The addition of 0.5 mM NH3 to the PFK buffer did not interfere with the PFK buffer pH.
Arioli S., Della Scala G., Martinović A., Scaglioni L., Mazzini S., Volonté F., Pedersen M.B, & Mora D. (2022). In Streptococcus thermophilus, Ammonia from Urea Hydrolysis Paradoxically Boosts Acidification and Reveals a New Regulatory Mechanism of Glycolysis. Microbiology Spectrum, 10(3), e02760-21.
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2

Measuring PFK Activity in Streptococcus thermophilus

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PFK activity was measured on the total protein extract of EdCs using the Phosphofructokinase (PFK) Activity Colorimetric assay kit (Sigma-Aldrich, Milan, Italy). To this aim, EdCs were prepared as described previously and subsequently subjected to total protein extraction. Mechanical cell disruption was carried out using a Precellys bead beater (Advanced Biotech Italia SRL, Seveso, Italy). To minimize the interference of NADH-generating enzymatic activities, the PFK assay was performed in the presence of 30 nM Br-Acivicin, a chemical inhibitor of glyceraldehyde-3-phosphate dehydrogenase (43 (link)). Several sample dilutions were tested to ensure that the readings were within the linear range of the standard curve. After this setup, S. thermophilus EdCs total protein extract diluted 1 to 200 in PFK buffer was used in the reaction. When necessary, NH3 or NH4Cl was added to the reaction mix to reach a final reaction concentration of 0.5 mM. The addition of 0.5 mM NH3 to the PFK buffer did not interfere with the PFK buffer pH.
Arioli S., Della Scala G., Martinović A., Scaglioni L., Mazzini S., Volonté F., Pedersen M.B, & Mora D. (2022). In Streptococcus thermophilus, Ammonia from Urea Hydrolysis Paradoxically Boosts Acidification and Reveals a New Regulatory Mechanism of Glycolysis. Microbiology Spectrum, 10(3), e02760-21.
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3

Phosphofructokinase Activity Assay

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PFK1 activity was measured using the Phosphofructokinase (PFK) Activity Colorimetric Assay Kit (Sigma) following the manufacturer’s recommendations.
McGrail K., Granado-Martínez P., Esteve-Puig R., García-Ortega S., Ding Y., Sánchez-Redondo S., Ferrer B., Hernandez-Losa J., Canals F., Manzano A., Navarro-Sabaté A., Bartrons R., Yanes O., Pérez-Alea M., Muñoz-Couselo E., Garcia-Patos V, & Recio J.A. (2022). BRAF activation by metabolic stress promotes glycolysis sensitizing NRASQ61-mutated melanomas to targeted therapy. Nature Communications, 13, 7113.
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4

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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5

Metabolic Enzyme Activities in Flight Muscle

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The PYK activity was measured using the Pyruvate Kinase Activity Colorimetric/Fluorometric Assay Kit (BioVision, K709). Approximately 50 mg of flight muscle tissue was homogenized with 200 µL assay buffer and incubated on ice for 10 min. The samples were then centrifuged for 10 min at 4°C at 14,000× g. The supernatant was diluted 100 times with the assay buffer for measurement. The values were normalized against lysate protein levels. The PFK activity was measured using the Phosphofructokinase (PFK) Activity Colorimetric Assay Kit (Sigma-Aldrich, MAK093). Approximately 50 mg of flight muscle tissue was homogenized with 500 µL ice-cold assay buffer and centrifuged for 20 min at 4°C at 14,000× g. The supernatant was diluted 50 times with the assay buffer for measurement. The values were normalized against lysate protein levels. The PDH activity was measured using the PDH Activity Colorimetric Assay Kit (BioVision, K697). Nearly 50 mg of flight muscle tissue was homogenized with 400 µL assay buffer and incubated on ice for 10 min. The samples were then centrifuged for 5 min at 4°C at 14,000× g. The supernatant was diluted 10 times with the assay buffer for measurement. The values were normalized against lysate protein levels.
Ding D., Zhang J., Du B., Wang X., Hou L., Guo S., Chen B, & Kang L. (2022). Non-canonical function of an Hif-1α splice variant contributes to the sustained flight of locusts. eLife, 11, e74554.
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