MAK093 is designed based on the indirect detection of phosphofructokinase activity. The ADP generated from the conversion of fructose-6-phosphate to fructose-diphosphate should react with a substrate and be converted to AMP and NADH. This process reduces a colorless probe to a colored product with strong absorbance at 450 nm. Therefore, considering that all three major isoforms in mammals, PFK-M (muscle), PFK-L (liver), and PFK-P (platelet), should catalyze the same enzymatic reaction producing ADP, it is assumed that this kit will not be selective for one specific isoform.
MAK093
Phosphofructokinase (PFK) Activity Colorimetric Assay Kit
sufficient for 100 colorimetric tests
Synonyme(s) :
PFK Activity Assay Kit
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A propos de cet article
NACRES:
NA.84
UNSPSC Code:
12161503
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sufficient for 100 colorimetric tests
detection method
colorimetric
relevant disease(s)
cancer; orthopedic diseases; aging/geriatric diseases
storage temp.
−20°C
Gene Information
human ... PFKL(5211), PFKM(5213), PFKP(5214)
mouse ... PFKL(18641), PFKM(18642), PFKP(56421)
rat ... PFKL(25741), PFKM(65152), PFKP(60416)
General description
Phosphofructokinase (PFK) catalyzes the third step of glycolysis, the conversion of fructose-6-phosphate to fructose-1,6-diphosphate, the rate limiting step of glycolysis. PFK activity is highly regulated by multiple cofactors in addition to post-translational modifications and can be used to measure glycolytic flux in tissues. Deficiencies in PFK activity can result in the glycogen storage disease, glycogenosis type VII (Tarui′s disease), which is a glycogen storage disease.[1][2]
Application
Suitable for the measurement of phosphofructokinase activity, analysis of glucose metabolism and cell signaling and screening anti-cancer drugs in animal tissues (liver, brain, heart, muscles etc.) and cell culture (adherent or suspension cells).
Phosphofructokinase (PFK) Activity Colorimetric Assay Kit has been used to measure the activity of phosphofructokinase.
Biochem/physiol Actions
The Phosphofructokinase Colorimetric Assay kit provides a simple and direct procedure for measuring PFK activity in a variety of samples. PFK activity is determined by a coupled enzyme assay, in which fructose-6-phosphate and ATP is converted to fructose-1,6-diphosphate and ADP by PFK. The ADP is converted by the enzyme mix to AMP and NADH. The resulting NADH reduces a colorless probe resulting in a colorimetric (450 nm) product proportional to the PFK activity present. One unit of PFK is the amount of enzyme that will generate 1.0 μmole of NADH per minute at pH 7.4 at 37 °C.
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Danger
Classe de stockage
10 - Combustible liquids
wgk
WGK 3
hcodes
Hazard Classifications
Aquatic Chronic 2 - ED ENV 1 - Resp. Sens. 1
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Does this kit work for all isoforms of Phosphofructokinase, such as PFK-M (muscle), PFK-L (liver), and PFK-P (platelet)?
1 answer-
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Is the standard curve created from the difference between the final and initial A_450 standard values, given the standards at final and initial time points? Alternatively, is the curve generated solely from the final A_450 standard values? Also, if we have fewer than 2x10^6 cells, how much assay buffer should be used for homogenization when the instructions state to homogenize 2x10^6 cells in 200uL of assay buffer?
1 answer-
The standard curve should be plotted after subtracting the 0 Standard reading from all Standard readings. Additionally, the PFK assay buffer suggested for sample preparation can be adjusted proportionally. For instance, 100 µl of PFK assay buffer can be used for 10 mg of tissue or 1 x 10^6 cells.
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Does the Positive Control in MAK093 include PFK-P, -L, or -M?
1 answer-
PFK-P (platelets), L (Liver), and M (Muscle) are all mammalian, and since the product is from bacillus sps, the question doesn't really apply to the PFK in use.
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