Therefore these techniques may be used for quantitation during protocol

Therefore these techniques may be used for

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Therefore, these techniques may be used for quantitation during protocol development. The immunoassay technique is also particularly suitable for screening large numbers of samples when a simple yes/no answer is required (e.g., when testing fractions from a chromatographic run). Assessing protein expression Suboptimal expression of the target protein can be addressed by various methods, based on the cause of the problem. If no target protein is detected in the extract, this may mean that the insert has been cloned in an incorrect reading frame. It is essential that the protein-coding DNA sequences are cloned in the proper translational reading frame in the vector. The best way to verify that the insert is in-frame is to sequence the cloning junctions. If yield of the target protein is low, it may be because the culture conditions have not been optimized for its expression. Investigate the effect of cell strain, medium composition, incubation temperature, and induction conditions (if applicable). Exact conditions will vary for each tagged protein expressed. With E. coli systems, analyze a small aliquot of an overnight culture by, for example, SDS-PAGE, and if available, use an activity assay. Generally, a highly expressed protein will be visible by Coomassie™ blue staining when 5 to 10 µl of an induced culture whose A 600 is ~1.0 is loaded on the gel. Nontransformed host E. coli cells and cells transformed with the parental vector should be run in parallel as negative and positive controls, respectively. The presence of the tagged protein in a total cell extract and its absence from a clarified lysate may indicate the presence of inclusion bodies. Check for inclusion bodies using light microscopy. They are often visible as dense spots in the cells. Refer to Chapter 8 for information on handling inclusion bodies. It is also worthwhile to check for expression by immunoblotting. Run an SDS-PAGE of induced cells and transfer the proteins to a nitrocellulose or PVDF membrane (such as Hybond™-C or Hybond-P). Detect tagged protein using anti-histidine or anti-GST antibody, as appropriate, or an antibody directed toward the specific target protein. Some tagged proteins may be masked on SDS-PAGE by a bacterial protein of approximately the same molecular weight. Immunoblotting can be used to identify tagged proteins in these cases. If the target protein is present in the post-lysate pellet, consider methods to enrich it. Alternatively, choose to secrete the product or add a stabilizing tag. If the target protein is adsorbed to cell debris, test extraction at varying ionic strengths and pH to dissociate it. Occasionally, a high basal level of expression is observed, and this may pose problems of its own (e.g., this is a major concern if the expressed protein is toxic). The cause may be a leaky promoter. Different vector systems rely on different constitutive and induced promoters, thus the most straightforward means of addressing this problem is to try another system. It is also possible that the vector is simply not compatible with the expression host; trying another vector or host should alleviate this problem.
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  • Spring '18
  • jeon sung jeong
  • ........., GE Healthcare

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