Rat Phosphofructokinase, Liver (PFKL) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to PFKL. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to PFKL. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After the TMB substrate solution is added, only those wells that contain PFKL, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of PFKL in the samples is then determined by comparing the O.D. of the samples to the standard curve.

For Use with serum, plasma, and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

Catalyzes the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate by ATP, the first committing step of glycolysis (By similarity). Negatively regulates the phagocyte oxidative burst in response to bacterial infection by controlling cellular NADPH biosynthesis and NADPH oxidase-derived reactive oxygen species. Upon macrophage activation, drives the metabolic switch toward glycolysis, thus preventing glucose turnover that produces NADPH via pentose phosphate pathway (By similarity).

GENE ID	25741
SWISS PROT	P30835
SYNONYMS	PFK-B; 6-phosphofructokinase type B; Phosphofructo-1-kinase isozyme B; Phosphohexokinase

Materials Supplied

Kit Components	96 Wells Quantity/Size
Pre-coated, ready-to-use 96-well strip plate	1 plate
Plate sealer for 96 wells	2
Standard	2 tubes
Diluent buffer	1 bottle
Detection Reagent A	1 bottle
Detection Reagent B	1 bottle
TMB Substrate	1 tube
Stop Solution	1 tube
Wash Buffer (30 ℅ concentrate)	1 tube
Product data sheet	1 copy

Storage

The TMB Substrate, Wash Buffer (30X concentrate), and the Stop Solution should be stored at 4°C upon receipt, while the other items should be stored at -20°C.

Performance Characteristics

REPEATABILITY	Intra-assay Precision (Precision within an assay): 3 samples with low, middle, and high-level PFKL were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level PFKL were tested on 3 different plates, with 8 replicates in each plate. CV(%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%
SENSITIVITY	The minimum detectable dose was 0.58ng/mL.
ASSAY RANGE	1.56-100ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of PFKL. No significant cross-reactivity or interference between PFKL and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between PFKL and all analogs, therefore, cross reactivity may still exist.