RFK ELISA Kit (Human)

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level Riboflavin Kinase (RFK) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level Riboflavin Kinase (RFK) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

Catalyzes the phosphorylation of riboflavin (vitamin B2) to form flavin-mononucleotide (FMN), hence rate-limiting enzyme in the synthesis of FAD. Essential for TNF-induced reactive oxygen species (ROS) production. Through its interaction with both TNFRSF1A and CYBA, physically and functionally couples TNFRSF1A to NADPH oxidase. TNF-activation of RFK may enhance the incorporation of FAD in NADPH oxidase, a critical step for the assembly and activation of NADPH oxidase.

The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Riboflavin Kinase (RFK) . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Riboflavin Kinase (RFK) . Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Riboflavin Kinase (RFK), 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 Riboflavin Kinase (RFK) in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Reconstitution & Storage Instructions Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Antibody Array (AA) Protocol

Component Amount Anti-RFK Microplate 96 Wells (12 x 8 Well Strips) RFK Lyophilized Standard 2 x 50 ng 100X Biotinylated RFK Detector Antibody 120 uL Avidin/HRP Conjugate 120 uL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL TMB Substrate 1 x 9 mL Stop Solution 1 x 6 mL Component Amount Component Amount Anti-RFK Microplate 96 Wells (12 x 8 Well Strips) Anti-RFK Microplate 96 Wells (12 x 8 Well Strips) RFK Lyophilized Standard 2 x 50 ng RFK Lyophilized Standard 2 x 50 ng 100X Biotinylated RFK Detector Antibody 120 uL 100X Biotinylated RFK Detector Antibody 120 uL Avidin/HRP Conjugate 120 uL Avidin/HRP Conjugate 120 uL Standard Diluent 1 x 20 mL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL 30X Wash Buffer 1 x 20 mL TMB Substrate 1 x 9 mL TMB Substrate 1 x 9 mL Stop Solution 1 x 6 mL Stop Solution 1 x 6 mL

This assay has high sensitivity and excellent specificity for detection of Riboflavin Kinase (RFK) . No significant cross-reactivity or interference between Riboflavin Kinase (RFK) and analogues was observed.