Description
Introduction: Myosin-light-chain kinase also known as MYLK or MLCK is a serine/threonine-specific protein kinase that phosphorylates the regulatory light chain of myosin II. These enzymes are important in the mechanism of contraction in muscle. Once there is an influx of calcium cations (Ca++) into the muscle, either from the sarcoplasmic reticulum or, more importantly, from the extracellular space, contraction of smooth muscle fibers may begin. First, the calcium will bind to calmodulin. This binding will activate MLCK, which will go on to phosphorylate the myosin light chain at serine residue 19. This will enable the myosin crossbridge to bind to the actin filament and allow contraction to begin (through the crossbridge cycle). Since smooth muscle does not contain a troponin complex like striated muscle does, this mechanism is the main pathway for regulating smooth muscle contraction.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to MLCK. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for MLCK and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain MLCK, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm +/- 2 nm. The concentration of MLCK in the samples is then determined by comparing the O.D. of the samples to the standard curve