Cell Signaling pGEM® β-Gal Control DNA Product pGEM® β-Gal Control DNA For Research Use Only. Not for Use in Diagnostic Procedures. Description: pGEM® β-Gal Control DNA contains the coding sequence of β-galactosidase downstream of an E. coli wildtype lacZ promoter. pGEM® β-Gal Control DNA can be used as a positive control in the E. coli S30 Extract System for Circular DNA. The wildtype lacZ promoter is not efficient for initiating transcription from a linear DNA template. Supplied as a 0.5mg/ml solution in TE buffer. Storage Conditions: Store at –20°C. Cell-Based Protein Expression Regulated Mammalian Expression System Product Regulated Mammalian Expression System Coumermycin A1 Novobiocin Sodium Salt Available Separately pReg neo Vector pF12A RM Flexi® Vector pF12K RM Flexi® Vector Size Cat.# Price (Fr) 1 system C9470 1058.00 5 mg C9451 145.00 1 g C9461 52.00 20 µg C9421 390.00 20 µg C9431 424.00 20 µg C9441 424.00 C9421, C9470, C9431, C9451, C9441 For Research Use Only. Not for Use in Diagnostic Procedures. C9461 For Research Use Only. Not for Use in Therapeutic or Diagnostic Procedures. Description: The Regulated Mammalian Expression System features low basal levels, robust and rapid induction, and downregulation of gene expression in mammalian cells. The Regulated Mammalian Expression System is based on a novel on/off switch that relies on the rapid and sensitive modulation by coumerin-related compounds of a chimeric transactivator protein. Nanomolar concentrations of the antibiotic coumermycin promote homodimerization of a chimeric transactivator that, in turn, binds to lambda operator sequences located upstream of a minimal promoter driving transcription of coding sequences for a protein of interest. The levels of protein expression can be regulated by adjusting the coumermycin concentration. More significantly, this expression can be promptly and effectively switched off by adding novobiocin, which acts as an antagonist by dissociating the dimerized transactivator protein. The protein coding region of interest is cloned into either the pF12A RM Flexi® Vector or pF12K RM Flexi® Vector, both of which are specially designed for Regulated Mammalian (RM) protein expression. These vectors incorporate regulatory promoter sequences upstream of the protein-coding region and are compatible with the Flexi® Vector System. In transient transfection paradigms, the pF12A or pF12K RM Flexi® Vector containing the protein-coding region of interest is co-transfected into mammalian cells together with the pReg neo Vector. The pReg neo Vector is designed to express a chimeric transactivator protein that interacts with the regulatory promoter region in the pF12A and pF12K RM Flexi® Vectors in a regulated fashion in response to coumermycin and novobiocin. Additionally, the pReg neo Vector encodes a neomycin phosphotransferase gene that allows stable cell selection and generation with the antibiotic G-418. Features: • Enhanced Data: High level of controlled induction combined with low basal protein expression. • Regulated Expression: Dose-response induction of protein expression; rapid and sensitive on/off switch for protein expression. • Versatility: Compatible with other Flexi® Vectors. Storage Conditions: Store at –20°C. Protocol Regulated Mammalian Expression System Technical Manual Part# TM289 For complete and up-to-date product information visit: www.promega.com/catalog 231 Size Cat.# Price (Fr) 20 µg L4731 187.00 Single Step (KRX) Competent Cells Product Single Step (KRX) Competent Cells L-Rhamnose Monohydrate Size Cat.# Price (Fr) 20 × 50 µl L3002 406.00 10 g L5701 68.00 50 g L5702 289.00 For Research Use Only. Not for Use in Diagnostic Procedures. Description: The Single Step (KRX) Competent Cells are designed for efficient transformation and tightly controlled protein expression. This one strain consolidates the best attributes of these two steps into one strain to evaluate protein expression in E. coli. Transformation efficiencies are greater than 108 cfu/μg, similar to other highly competent cells. The single step cells are available in single transformation size (50μl). KRX also can be used for blue/white screening. Single Step (KRX) is an E. coli K strain that contains a chromosomal copy of the T7 RNA polymerase driven by a rhamnose promoter (rhaBAD) to provide dramatic control of the proteins expressed via a T7 promoter. Pre-induced expression protein levels are significantly lower than those of BL21(DE3)derived strains. This feature facilitates cloning and expression of proteins toxic to E. coli. Genotype: [F´, traD36, ΔompP, proA+B+, lacIq, Δ(lacZ)M15] ΔompT, endA1, recA1, gyrA96 (Nalr), thi-1, hsdR17 (rk–, mk+), e14– (McrA–), relA1, supE44, Δ(lac-proAB), Δ(rhaBAD)::T7 RNA polymerase. Features: • Save Time: In two days, you can transform your vector into the Single Step (KRX) Competent Cells and be ready for protein expression. • Controlled Protein Expression: For overall expression of cloned proteins, the Single Step (KRX) Competent Cells provide dramatic control of expressed protein-coding regions. • Achieve High Yields: Protein expression levels were shown to be as high as or higher than levels expressed in BL21(DE3)-derived strains. Protocol Single Step (KRX) Competent Cells Technical Bulletin Part# TB352 13 Protein Expression and Analysis Pagina 234
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