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Therefore, these techniques may be used for quantitation during protocol development. Theimmunoassay technique is also particularly suitable for screening large numbers of samples whena simple yes/no answer is required (e.g., when testing fractions from a chromatographic run).Assessing protein expressionSuboptimal expression of the target protein can be addressed by various methods, based on the causeof the problem. If no target protein is detected in the extract, this may mean that the insert has beencloned in an incorrect reading frame. It is essential that the protein-coding DNA sequences are clonedin the proper translational reading frame in the vector. The best way to verify that the insert is in-frameis to sequence the cloning junctions.If yield of the target protein is low, it may be because the culture conditions have not been optimizedfor 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. colisystems, analyze a small aliquot of an overnight culture by, for example, SDS-PAGE, and ifavailable, use an activity assay.Generally, a highly expressed protein will be visible by Coomassie™ blue staining when 5 to 10 µl of aninduced culture whose A600is ~1.0 is loaded on the gel. Nontransformed host E. colicells and cellstransformed 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 mayindicate the presence of inclusion bodies. Check for inclusion bodies using light microscopy. They areoften 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 andtransfer the proteins to a nitrocellulose or PVDF membrane (such as Hybond™-C or Hybond-P). Detecttagged protein using anti-histidine or anti-GST antibody, as appropriate, or an antibody directed towardthe specific target protein. Some tagged proteins may be masked on SDS-PAGE by a bacterial proteinof approximately the same molecular weight. Immunoblotting can be used to identify tagged proteinsin 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 todissociate 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. Differentvector systems rely on different constitutive and induced promoters, thus the most straightforwardmeans of addressing this problem is to try another system. It is also possible that the vector is simplynot compatible with the expression host; trying another vector or host should alleviate this problem.