Horseradish peroxidase (HRP) labeled goat anti-rabbit IgG application: WB (1:1000 - 1:50000), ELISA (1:500 - 1:10000) IHC (1:50 - 1:500). Because the sensitivity of the detection method in specific experiments, temperature and incubation time can affect the optimal working concentration of the antibody, the experimenter needs to optimize the optimal concentration according to the specific conditions when conducting specific experiments. Reactivity: The goat anti-rabbit secondary antibody specifically reacts with rabbit IgG molecules and cross-reacts with other rabbit immunoglobulins (including IgM, IgE, IgD, IgA, etc.). The secondary antibody does not cross-react with the non-immunoglobulin in the serum, but may cross-react with immunoglobulins of other species.
Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG hrp excess leads to the principle of ecl quenching during wb experiment: quenching is caused by excessive amount of hrp enzyme in the color reaction, the recommended improvement method Yes, reducing the amount of hrp, including reducing the amount of protein loaded, reducing the concentration of primary antibody, reducing the concentration of secondary antibody, in short, reducing the amount of hrp binding on the membrane. Some people may ask, I can't do a lot of antibody or protein loading, but I can reduce it instead. How is this possible? Oh, in fact, we must first figure out who is over-exposed in the color reaction, of course, the substrate, because the brightness is related to the amount of hrp, hrp is related to the amount of protein of interest, only the amount of substrate excess hrp can Clearly react to the amount of protein of interest (the qualitative experiment is not considered here). Therefore, hrp must be insufficient relative to the substrate. Secondly, there should be many friends who have encountered such a phenomenon: when exposed, the strip is particularly bright, but if the film is pressed once or twice, the strip will be destroyed, but if the film is washed again with TBST, then De-exposure, the fluorescence can last longer, which is because the washing, so that some of the original combination of weak secondary antibodies fell, the amount of hrp decreased when exposed, will not consume the substrate faster, so The time of illuminating becomes longer.
Finally, how to save the labeled goat anti-rabbit IgG? Expert advice: Keep at -20 ° C. The shelf life is at least 1 year after receipt of the goods, the appropriate preservation method can extend the shelf life. Recommended reading" TRITC labeled secondary antibody supply http://
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