By Rocky S. Tuan
Top researchers and specialists current wide-ranging equipment for detecting and keeping apart expressed gene products-recombinant proteins. those cutting-edge concepts describe various 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 task, in addition to chapters on in situ detection of gene expression. while mixed with a better half quantity by way of a similar editor, Recombinant Gene Expression Protocols, either volumes advisor the examine employee during the complete trip of recombinant gene expression.
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Additional info for Recombinant Protein Protocols: Detection and Isolation (Methods in Molecular Biology Vol 63)
Sample text
20 @, cell extract (seeNote 6). b. 8. c. 1 pCi of [i4C]chloramphenicol. 1 U of bacterial CAT (positive control) and reaction buffer (negativecontrol). 2. Start the reaction by adding 20 pL of 4 nnJ4acetyl CoA (see Note 7) to each reaction. Incubateat 37°C for 1 h (seeNote 3). 3. Stopthe reactionand extractthe chloramphenicolwith 1mL of cold ethyl acetate (4°C) by vortexing for at least 30 s. 35 CAT Reporter Enzyme Activity 4 5. 6 7 8 9 10 11. 12. 7-mL mlcrofuge tubes Dry samples m a Savat Speed Vat Concentrator Resuspend pellets m 30 pL of cold ethyl acetate With a #2 pencil, draw a lme about 2 cm from one edge of the TLC plate to mark the starting lme.
Flow diagram for expression and detection of SEAP reporters in transfected cells. Kain 52 Nhe I (29) Xho 1134) B I II 138) Hifl % Ill (48) Sph I(611 I IO 20 30 40 50 IEAP ~~~GGGA~GAGCTC~TACGCGTGC~A~CT~G&TAA~TA&TKW Am cm Nhel Xhol By/II //hKlIII ~ SphI xi-Asp718 I KpnI Fig. 2. The MCS (shown below the plasmid map) allows promoter DNA fragments to be inserted upstream of the SEAP gene. Enhancerscan be cloned into either the MCS or unique downstream sites. Unique restriction sites are in boldface on the plasmid map.
Unique restriction sitesare in boldfaceon the plasmid map. The MCS is shownbelow the plasmid map. The sequenceof pSEAP-Control hasbeendeposited in GenBank (Accession# U09661). early promoter) downstream. pSEAP-Control should express SEAP in most cell types and provides an important positive control for transfection and expressionof exogenousDNA. The map of the pSEAP-Control vector is shown in Fig. 3. pSEAP-Enhancer contains the SV40 enhancerinserted downstream of the SEAP gene in pSEAP-Basic. pSEAP-Enhancer is intended for analyzing promoter sequences inserted into the MCS.