PrecisionAb Antibodies were chosen for this multiplex western blot because of their superior performance and reproducibility, having been rigorously tested and selected for high specificity, high sensitivity, and consistency. Composite blot image for quadruple western blotting.įigure 1 shows a composite blot image with four distinct proteins visualized using StarBright Blue 520 and StarBright Blue 700 Secondary Antibodies, in combination with Dylight 800 and a rhodamine labeled recombinant human hFAB antibody, for the detection of popular housekeeping proteins on the ChemiDoc MP Imaging System. They are excited with blue light (440–470 nm) and are available in two colors: StarBright Blue 520 emits in the green range of the spectrum (emission maximum: 520 nm) and StarBright Blue 700 maximally emits in the far red/near infrared region of the spectrum (700 nm).įig. StarBright Dyes’ fluorescence emission is restricted to a narrow spectral band, making them ideal for multiplex fluorescence imaging for western blotting. Labeled with exceptionally bright and stable semiconducting nanoparticles. Read the introductory guide for detailed information on multiplex fluorescent western blotting. Imaging with ChemiDoc MP Imaging System.hFAB Rhodamine Anti-Housekeeping Protein Primary Antibodies.Fluorescent StarBright Blue 700 and 520 Secondary Antibodies.Greater consistency of readout using fluorescent dyes rather than the variability of an enzymatic reactionĪmount of information collected is maximized, using as little of your rare/precious sample as possibleīio-Rad offers a new generation of multiplex western blotting reagents and equipment, that includes: Results in context by simultaneous analysis of several proteins and parameters Multiple samples are analyzed under the same experimental conditions The benefits of fluorescent western blotting multiplexing are: By optimizing your experiments to include fluorescent multiplexing, you can avoid the errors commonly associated with stripping, reprobing, and cutting blots and ensure you are publishing quantifiable, reproducible results.The increasing need to maximize data from a single sample has led to the development of fluorescent based multiplexing assays. Multiplex fluorescent western blotting enables the simultaneous analysis of multiple proteins in a single sample on the same blot, obtaining as much information as possible in one experiment. Multiplexing also allows you to perform accurate analysis when there are only small shifts in molecular weight, e.g., post-translational modifications. ![]() This will save you time and sample and allow you to answer more complex questions. An additional benefit of the chemistry is blots can be cataloged for extended periods of time when stored properly.īy combining different antibody species with fluorophores of varying wavelengths, you can use fluorescent western blotting to create multiplex experiments. Furthermore, method development is simplified due to the linearity of the reaction. Unlike traditional western blotting detection methods, fluorescence is not subject to enzyme kinetics making quantitation more reproducible. The basic principle of the technique is that the level of fluorescence emitted by a fluorophore conjugated to the detection antibody is directly related to the level of protein expression. Fluorescent western blotting is a method that is increasing in popularity because it addresses the need for accurate, quantitative determination of protein expression.
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