![]() Refer to Table 1 to identify the most appropriate HRP chemiluminescent substrate based on the abundance of your target protein of interest, abundance of sample containing the target protein, and the level of sensitivity and type of instrumentation available for detection. Several varieties of Thermo Scientific Pierce ECL and SuperSignal chemiluminescent HRP substrates are available that provide different levels of sensitivity for chemiluminescence western blotting. Because its reaction rate remains linear, detection sensitivity can be improved by simply allowing a reaction to proceed for a longer time period.Īs HRP is the most popular enzyme used in western blotting it will be discussed throughout this article as our example. As a label for Western blotting, AP offers a distinct advantage over other enzymes. AP has optimal enzymatic activity at a basic pH (pH 8–10) and can be inhibited by cyanides, arsenate, inorganic phosphate and divalent cation chelators, such as EDTA. Alkaline phosphatase (AP) catalyzes the hydrolysis of phosphate groups from a substrate molecule resulting in a colored or fluorescent product and the release of light as a byproduct of the reaction. ![]() Because of the relatively small size of the HRP enzyme, further increases in sensitivity may be achieved by using poly-HRP conjugated secondary antibodies and may eliminate the need for using ABC-type amplification systems for some researchers. In addition, its high turnover rate, good stability, low cost, and wide availability of substrates makes HRP the enzyme of choice for most applications. Antibody-HRP conjugates are superior to antibody-AP conjugates with respect to the specific activities of both the enzyme and antibody. HRP functions optimally at a near-neutral pH and can be inhibited by cyanides, sulfides and azides. gondii RH strain tachyzoites (P < 0.005), and their survival time increased significantly compared to the negative control.Horseradish peroxidase (HRP) catalyzes the oxidation of substrates by hydrogen peroxide, resulting in a colored or fluorescent product and the release of light as a by-product of the reaction. Finally, vaccinated mice were significantly protected against lethal challenge with live T. These results show that the chimeric protein recombinant SAG1/2 can elicit a Th1-associated protection against T. Specific production of IFN-gamma, the Th1-type cytokines, was also found in stimulated splenocytes from vaccinated mice. ![]() Significant proliferation of splenocytes stimulated with tachyzoite total protein lysate was observed in vaccinated BALB/c mice but not in those from negative control mice. Mice immunized with the recombinant SAG1/2 reacted specifically with the native SAG1 and SAG2 of T. To further investigate the immunological characteristic of the recombinant protein, the recombinant SAG1/2 was injected subcutaneously into BALB/c mice, and their serum was tested against total protein lysate of T. pastoris-derived recombinant SAG1/2 was sensitive and specific and suitable for use as antigen for detecting anti-Toxoplasma antibodies. Results of Western blots targeted at Toxoplasma IgG and IgM showed that the recombinant SAG1/2 reacted with all sera from the toxoplasmosis cases but none with the Toxoplasma-negative serum samples. Eighty human serum samples, including 60 from confirmed cases of toxoplasmosis, were tested against the purified recombinant SAG1/2 in Western blots. In this study, we successfully expressed a chimerical surface antigen 1 and 2 (SAG1/2) of Toxoplasma gondii in Pichia pastoris. ![]()
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