Download Recombinant Protein Protocols: Detection and Isolation by Rocky S. Tuan PDF

By Rocky S. Tuan

Prime researchers and specialists current wide-ranging equipment for detecting and separating expressed gene products-recombinant proteins. those state of the art concepts describe quite a few molecular tags and labels, together with enzymes, ligand-binding moieties, and immunodetectable molecules. There also are the way to observe interactive proteins and gene expression-mediated changes in mobile job, in addition to chapters on in situ detection of gene expression. while mixed with a spouse quantity by means of a similar editor, Recombinant Gene Expression Protocols, either volumes consultant the learn employee throughout the complete trip of recombinant gene expression.

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Extra info for Recombinant Protein Protocols: Detection and Isolation (Methods in Molecular Biology Vol 63)

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Cockmg (1992) Quantlfication and comparison of chloramphemcol acetyltransferase activity m transformed plant protoplasts using high-performance liquid chromatography- and radiolsotope-based assays. Anal Blochem 210,87-93. , Merkelbach, S , Gehlen, J , and Fladung, M (1993) The nonradloactive chloramphemcol acetyltransferase-enzyme-linked immunosorbent assay test IS suited for promoter actlvlty studies in plant protoplasts Anal. Biochem 211, 113-116. 10. Nielson, D. , and Shapiro, D. J. (1989) A highly sensltlve, mixedphase assay for chloramphenicol acetyltransferase activity m transfected cells Anal Biochem 179, 19-23.

Samples with more than 30% conversion should be assayed again wtth different dilutions of the cell extracts The senstttvtty of the assay IS about 1Cr2 CAT units Its lmear response range IS between 1W2 to 4 x IO-’ CAT units (8) Several assay systems have been developed to avoid the need for radiolabeled substrate. Besides BodlpyTM chloramphemcol derivative, one modification with srmtlar sensmvlty to the TLC-based assay is an assay based on HPLC separation (2,8) An alternative method is the enzyme-linked mununosorbance assay using CAT-specific antibodies with comparable limit of detection to that of the radioactive tests (Boehringer Mannheim, Mannheim, Germany; 9).

A secondpolyadenylation signal upstream of the MCS reduces background transcription (7). The vector backbonecontains an fl origin for singlestrandedDNA production and a pUC 19 origin of replication and an ampicillin-resistance gene for propagation in E. coli. The sequenceof pSEAP-Basic hasbeen depositedin GenBank(Accession# UO9660). ing of the SEAP transcript in eukaryotic cells. A second polyadenylation signal upstream of the MCS reduces background transcription (6). The vector backbone also contains an fl origin for single-stranded DNA production, a pUC 19 origin of replication, and an ampicillin resistancegene for propagation in Escherichia coli.

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