If further denaturing is required a reducing agent (DTT or Beta mercaptoethanol) may be used to disrupt S-S bonds. As such their use with antibodies raised against denatured forms or synthetic peptides of the target protein is often recommended. Ionic detergents are considered harsher than the non-ionic detergents because of their ability to interfere with protein structure. The best known of the ionic detergents is sodium dodecyl sulfate (SDS) while non-ionic detergents include Tween, Triton X and NP-40. The primary active agent in lysis buffer is the detergent which can be ionic or non-ionic. Lysis is the process in which the cell membrane is broken and the contents of the cell are re-suspended in a soluble form. There are several steps for maximising the recovery of your target protein from the sample. Initial improvements in the detection and quantification of low abundant proteins can be made at the sample preparation stage. As such detection of proteins with low expression levels is a common problem for researchers. To help you optimize both the detection and quantification of such proteins and overall quality of the final western blot image, we have put together useful tips for the following steps: The technique of western blotting illuminates molecular events including protein expression, protein localisation, protein-protein interactions or post translational modifications (PTMs). These molecular events are often very subtle. For example within an individual signaling pathway only a certain percentage of the molecules may have undergone a PTM. Enhance detection and quantification of low abundance protein
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