Science Catalog 2012 Life Cell Signaling BL21(DE3)pLysS Competent Cells Product BL21(DE3)pLysS Competent Cells, >106cfu/µg For Research Use Only. Not for Use in Diagnostic Procedures. Size Cat.# Price (Fr) 1 ml L1191 340.00 Description: BL21(DE3)pLysS Competent Cells allow high-efficiency protein expression of any gene that is under the control of a T7 promoter and has a ribosome binding site. BL21(DE3)pLysS is lysogenic for λ-DE3, which contains the T7 bacteriophage gene I, encoding T7 RNA polymerase under the control of the lac UV5 promoter. BL21(DE3)pLysS also contains a plasmid, pLysS, which carries the gene encoding T7 lysozyme. T7 lysozyme lowers the background expression level of target genes under the control of the T7 promoter but does not interfere with the level of expression achieved following induction by IPTG. One milliliter is sufficient for ten transformations. Genotype: F–, ompT, hsdSB (rB–, mB–), dcm, gal, λ(DE3), pLysS, Cmr. Features: • T7 Promoter Expression: Contains an IPTG-inducible gene for T7 RNA polymerase. • Convenient: A time-saving alternative to making competent cells. Storage Conditions: Store at –70°C. Protocol E. coli Competent Cells Technical Bulletin A. 100 120 140 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000 10,000 0 BL21(DE3) BL21(DE3) pLysS B. 10,000 1,700 1,000 100 14 10 1 8 Rosetta™ 2 pLysS KRX 20 40 60 80 0 Pre-Induction Expression Part# TB095 Mass Spectrometry Analysis ProTEV Plus Product ProTEV Plus Size Conc. Cat.# Price (Fr) 1,000 u 5 u/µl V6101 190.00 8,000 u 5 u/µl V6102 1140.00 For Research Use Only. Not for Use in Diagnostic Procedures. Description: ProTEV Plus is an engineered form of TEV protease, a highly specific proteolytic enzyme that cleaves within a seven-amino-acid sequence. It can be used to cleave protein fusions that have been engineered to contain the seven-amino-acid sequence at the desired cleavage site. ProTEV Plus also contains an HQ tag located at the N-terminus of the protein, which allows it to be immobilized on affinity resins and removed from the cleavage reaction. Features: • Active Over a Wide Range of pH and Temperatures: Cleave individual fusion proteins using optimal conditions to maintain activity and correct conformation. • HQ-Tagged: Convenient removal of ProTEV Plus using Ni-based affinity resins after cleavage. • Cleaves Fusion Proteins Directly in Solution or Immobilized on Affinity Resins: ProTEV Plus is easy to use in multiple experimental formats. Storage Conditions: Store at –20°C. Protocol ProTEV Plus Protease Product Information Part# 9PIV610 Product ProteaseMAX™ Surfactant, Trypsin Enhancer Size Cat.# Price (Fr) ProteaseMAX™ Surfactant, Trypsin Enhancer 1 mg V2071 95.00 5 × 1 mg V2072 404.00 For Research Use Only. Not for Use in Diagnostic Procedures. Description: ProteaseMAX™ Surfactant, Trypsin Enhancer, is designed to improve in-gel and in-solution protein digestion. ProteaseMAX™ Surfactant ensures fast and efficient protein digestion with proteases such as Trypsin, Chymotrypsin and Lys-C. For in-gel protein digestion, ProteaseMAX™ Surfactant offers time and labor savings. Digestion step is complete in 1 hour, and the surfactant provides concurrent extraction of peptides from gels, eliminating the need for post-digestion peptide extraction. The surfactant also improves recovery of longer peptides that are retained in the gel under a standard extraction protocol. For additional data, refer to scientific posters PS094 and PS099. For in-solution digestions, ProteaseMAX™ Surfactant solubilizes proteins, including difficult proteins (i.e., membrane proteins), and enhances protein digestion by providing a denaturing environment prior to protease addition. 43 ProteaseMAX™ Surfactant degrades over the course of a digestion reaction, yielding products that are compatible with downstream methods such as mass spectrometry (MS) and liquid chromatography (LC). No long-term negative effect of the residual surfactant on the ion optics and capillary of mass spectrometers has been observed. ProteaseMAX™ Surfactant can be used with existing in-gel or in-solution digestion protocols. Features: BL21(DE3) BL21(DE3) pLysS Rosetta™ 2 pLysS KRX an optical density (O.D.600) of 0.8–1.0 and then moved to a 25°C incubator shaker. When cultures reached an O.D.600 of 1.0–1.5, protein expression was induced using either 0.1% rhamnose or 1mM IPTG and grown overnight Pre-induction and post-induction expression levels of firefly luciferase. Cells were transformed with the pF1K T7 Flexi® Vector containing the firefly luciferase gene. Cultures were grown at 37°C to at 25°C. Samples for luciferase assays were removed prior to and after induction. Panel A. Firefly luciferase expression level was determined using the Bright-Glo™ Luciferase Assay Reagent. Pre- and post-induction firefly luciferase expression levels were normalized to cell number (n = 3). Panel B. Induction ratios were calculated by dividing the post-induction luminescence values by the pre-induction values. 232 • No Requirement for Peptide Extraction following In-Gel Digestions: Increase the number of samples processed. • One-Hour Digestion: Save time by avoiding overnight digestions. • Improved Peptide Recovery from Gels: Increase in protein sequence coverage, thus increasing confidence of protein identification. • Enhanced Protein Solubilization: Solubilize complex proteins such as membrane proteins at room temperature, avoiding high temperatures and preventing precipitation. • Self-Degradable: Use directly for mass spectrometry analysis without additional inactivation steps such as heating or acid treatment. Storage Conditions: Store lyophilized ProteaseMAX™ Surfactant at –20°C. Protocol ProteaseMAX™ Surfactant, Trypsin Enhancer Technical Bulletin Part# TB373 For complete and up-to-date product information visit: www.promega.com/catalog Induction Ratio Normalized luminescence (RLU/OD) Normalized luminescence (RLU/OD) 032MA KRX Roset t a™ 2 pLysS BL21(DE3) BL21(DE3) pLysS Pagina 235
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