Most assays are carried out for a fixed period time (end point assays) and the reaction is halted by the addition of a stop reagent (e.g. acid). However, in continuous assays the appearance of product (less commonly the consumption of substrate) is recorded continuously (e.g. by means of a chart recorder).
The main difference between the two methods is how measurements are taken. In the endpoint assay, a final measurement is taken to measure the total amount of substrate/products. In the kinetic assay method, multiple measurements are taken over the course of the reaction.
Enzyme assays can be split into two groups according to their sampling method: continuous assays, where the assay gives a continuous reading of activity, and discontinuous assays, where samples are taken, the reaction stopped and then the concentration of substrates/products determined.
What are the advantages and disadvantages of using a chemiluminescent reaction versus a colored precipitate reaction. - Chemiluminescent is expensive, but extremely sensitive and can be exposed as many times as needed. - Colored precipitation is inexpensive , but less sensitive and once overexposed is ruined.
The key difference between kinetic and end point reaction is that in kinetic reaction method, we measure the difference in absorbance between two points during the progression of the reaction whereas, in end point reaction method, we measure the total amount of analytes that participate in the reaction.
The kinetic method requires no enzymes, has no lag phase, and has good sensitivity. A major advantage of the reaction is that it occurs at a temperature of 37 °C or lower. The results obtained by all three methods agree well with those for a continuous-flow procedure in which diacetyl is a reagent.
Most enzyme assays are based on spectroscopic techniques, with the two most commonly used being absorption and fluorescence Fersht (1999). The wavelength used for following the reaction rate should be one that yields the greatest difference in absorption between the substrate and the product.
Enzyme activity is the amount of substrate converted by the enzyme in moles per unit time. It measures the amount of active enzyme present in a mixture at a given time. On the other hand, specific activity is the activity of the enzyme per mg of total enzyme. It measures the purity of the enzyme in a mixture.
The Lineweaver–Burk plot was widely used to determine important terms in enzyme kinetics, such as Km and Vmax, before the wide availability of powerful computers and non-linear regression software. The y-intercept of such a graph is equivalent to the inverse of Vmax; the x-intercept of the graph represents −1/Km.
In addition to enabling multiplexing, fluorescent western blot detection has several other advantages compared to enzyme-based chemiluminescent substrate detection. Fluorescent western blotting provides accurate, quantitative results, stable signals, and the ability to conserve sample due to multiplexing.
Fluorescent detection can be less sensitive than chemiluminescent detection; chemiluminescent Westerns can be 10-100 times more sensitive, depending on the protein in question.
what is the main difference between staining and immunological detection? - Staining is not specific, it will bind all proteins. and shows purity Staining uses dyes. (Coomassie Blue, Ponceau S)- Immunological (western blot) detection is specific (one protein) because antibodies will bind to epitope tag on antigen.