By Rocky S. Tuan
Major researchers and specialists current wide-ranging tools for detecting and separating expressed gene products-recombinant proteins. those cutting-edge concepts describe a number of molecular tags and labels, together with enzymes, ligand-binding moieties, and immunodetectable molecules. There also are how you can 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 significant other quantity through a similar editor, Recombinant Gene Expression Protocols, either volumes consultant the examine employee throughout the complete trip of recombinant gene expression.
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Extra resources for Recombinant Protein Protocols: Detection and Isolation (Methods in Molecular Biology Vol 63)
4. Transfer the supematants to fresh Eppendorf tubes. These samples can be stored at -20°C mdefimtely 5. In an Eppendorf tube, add 100 pL of 2X SEAP buffer to 100 pL (or an emplritally determmed dllutlon) of sample As a zero standard, make up one mix substltutmg the sample with water 6. MIX by vortexmg 7 Transfer the contents of each tube (for example, 190 &) to the well of a flat bottomed 96-well microtlter plate, takmg care to avoid creating an bubbles. 8 Prewarm by incubating the plate at 37°C for 10 min 9 During this Incubation, make up the p-nitrophenol phosphate (the substrate) solution and prewarm it at 37°C 10.
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. Spot 5 mL of the samples at a time on the lure To mmlmlze the size of the spot, dry each spot before spotting another 5 mL until the samples are used up Allow at least 1 5-cm space between two sampies Dry completely Run samples on the TLC plates in a chamber pre-equilibrated with chloroformmethanol (90:10, ascendmg) about l-cm deep.
A general purpose plasmld vector, pBC12/PL/SEAP (6), that provides From Methods m Molecular Bfology, vol 63 Recombrnant Protern Protoco/s Detechon and /so/at/on Edtted by R Tuan Humana Press Inc , Totowa. NJ 47 42 Bates and Malim rat preproinsulin II gene (intron and polyadenylation) Fig. 1. Plasmid map of pBC12/PL/SEAP: The positions of the multiple cloning site, SEAP gene (solid box), rat preproinsulin II sequences(gray box), sequencesfor selection and propagation in bacteria (open box), and SV40 origin of replication (speckledbox) are indicated.