Colorful choices for your bright ideas

Transcription

1 Living Colors Fluorescent Proteins Reporters and Antibodies for Every Application Colorful choices for your bright ideas

2 Living Colors Fluorescent Proteins Full color spectrum cyan to far red Ideal for real-time and multicolor imaging Optimal fluorescence and photostability Extensive array of vector formats Suitable for a range of applications Specific fluorescent protein antibodies Easily detected and tolerated in live cells Clontech s Full Collection of Living Colors Fluorescent Proteins Fluorescent Protein Color Excitation Maximum (nm) Emission Maximum (nm) Relative Quantum Yield a Extinction Coefficient (M 1cm 1) b Brightness c Recommended Filter Set(s) d mplum far red ,000 4,100 C.T E2-Crimson far red ,000 28,980 C.T mraspberry far red ,000 12,900 C.T HcRed1 far red , C.T ; Omega XF102-2 mcherry red ,000 15,840 C.T ; Omega XF102-2 mstrawberry red ,000 26,100 C.T & AsRed2 red ,000 12,810 C.T & DsRed-Monomer red ,300 3,822 C.T c & DsRed2 red ,800 24,090 C.T c & tdtomato red ,000 95,220 C.T c & DsRed-Express red ,800 14,872 C.T c & DsRed-Express2 red ,600 15,000 C.T c & morange orange ,000 48,990 C.T morange2 orange ,000 34,800 C.T mbanana yellow ,000 4,200 C.T & ZsYellow1 yellow ,000 13,000 C.T & ZsGreen1 green ,000 39,130 C.T & AcGFP1 green ,500 26,650 C.T & Dendra2 (converted) Timer (converted) PAmCherry (activated) green (red) green (red) (red) 490 (553) 483 (558) 404 (564) 507 (573) 500 (583) (595) 0.50 (0.55) ND e (ND) (0.46) 45,000 (35,000) ND e (ND) 6,500 (18,000) 22,500 (19,250) ND e (ND) (8,280) C.T & (C.T c & 42005) C.T & (C.T c & 42005) C.T AmCyan1 blue ,000 29,250 C.T & EYFP g yellow ,000 24,300 EGFP g green ,000 16,100 ECFP g blue ,000 3,000 a The Relative Quantum Yield of each fluorescent protein (Q FP ) was determined using the following equation: Q FP = (Q fluo * F FP * OD fluo ) / (F fluo * OD FP ), where F is the fluorescence and OD is the optical density of either the fluorescent protein (FP) or the reference fluorophore (fluo). b The Molar Extinction Coefficient (e) for each protein was determined as follows: A = ecl (Beer-Lambert law) or e = A/c, where A = absorbance, c = concentration (determined by the Bradford Protein Assay), l = path length (1 cm). Each molar extinction coefficient was determined at the absorption maxima of its respective protein. c Brightness = (Quantum Yield)(Extinction Coefficient) d C.T. = Chroma Technologies e ND = not determined f NA = not applicable g For reference only; not sold by Clontech

3 Applications of Living Colors Fluorescent Proteins Clontech s fluorescent proteins are available in plasmid, lentiviral, and retroviral vector formats for a range of key applications. Visit for a complete list of our vectors. Fusions FRET Cellular & subcellular labeling Gene expression Kinetic studies Cell capture Cell cycle reporters Proteasome activity analysis Promoter reporters Timed gene expression In vivo imaging Stem cell applications Selected Applications of Living Colors Fluorescent Proteins Application Recommended Products Comments Protein localization Fusion fluorescent proteins from far red to cyan are available in a variety of vector formats for both N- and C-terminal fusions. AcGFP1, DsRed-Monomer, and mcherry proteins are ideal for fusions, since they are monomeric and so are least disruptive to the function of the protein of interest. Bacterial expression Subcellular labeling In vivo imaging Fluorescence resonance energy transfer (FRET) Protein dynamics & half-life We offer a variety of basic fluorescent protein vectors for bacterial expression. AcGFP1 DsRed-Monomer DsRed2 mcherry HcRed1 mplum E2-Crimson HcRed tdtomato AcGFP1 & DsRed-Monomer AcGFP1 & mcherry morange & mstrawberry morange & mcherry PAmCherry fluoresces when activated by nm light. Dendra2 converts from green to red when exposed to UV or blue light. The bacterial expression of fluorescent proteins is driven by the lac promoter. Available vector formats allow targeting of fluorescent proteins to the golgi apparatus, actin filaments, plasma membrane, nuclei, microtubules, endoplasmic reticuli, mitochondria, peroxisomes, and endosomes (Figure 1). Red and far red fluorescent proteins are ideal for in vivo imaging due to their reduced autofluorescence. Our td Tomato fluorescent protein is very bright and ideal for in vivo imaging (Figure 2). FRET can be used to monitor the proximity and interaction of two proteins. Success of FRET relies on the correct pairing of donor and acceptor fluorescent proteins. Photoactivatable and photoconvertible fluorescent proteins are powerful tools for studying protein half-life and trafficking, because they allow the selective marking of molecules present at the time of activation/ conversion. A B A tdtomato B GFP Figure 1. Subcellular labeling. AcGFP1 and DsRed2 protein fusions were transiently transfected and visualized by fluorescence microscopy. Panel A. pacgfp1-mito (mitochondria) and pdsred2-nuc (nucleus) in HEK 293 cells. Panel B. pacgfp1-golgi (Golgi apparatus) and pdsred2-nuc (nucleus) in HEK 293 cells. Figure 2. In vivo Imaging. False-color overlay images (regions of interest encircled) highlight tdtomato fluorescence, but not GFP fluorescence, in the cadaver model. Panel A. Implanted tube with 100 x 10 6 MDA-MB-231-tdTomato-expressing cells, imaged with the DsRed filter set. Panel B. Implanted tube with 100 x 10 6 MDA-MB-231-GFP-expressing cells, imaged with the GFP filter set. Exposure times: 1 sec.

4 Antibodies Clontech offers highly specific antibodies for the detection of our Living Colors fluorescent proteins. Our antibodies are compatible with many applications, including Western blotting, immunoprecipitation, and immunocytochemistry. EGFP Monoclonal Full-Length ZsGreen Polyclonal A.v. Monoclonal (JL-8) Full-Length GFP Polyclonal A.v. Peptide GFP Monoclonal Proven Uses a WB, IP, IC WB WB, IP, IC WB WB, IP, IC WB, IP, IC AcGFP1 ZsGreen1 GFP GFPuv EGFP b EYFP b ECFP b a WB = Western Blot, IP = immunoprecipitation, IC = immunocytochemistry b Not sold by Clontech Selection Guide for Living Colors Fluorescent Proteins DsRed Monoclonal DsRed Polyclonal mcherry Monoclonal Anti-RCFP Polyclonal Pan Proven Uses a WB WB, IP, IC WB WB mplum E2-Crimson mraspberry HcRed1 mcherry mstrawberry PAmCherry AsRed2 DsRed-Monomer DsRed2 tdtomato DsRed-Express DsRed-Express2 morange morange2 mbanana ZsYellow1 Timer ZsGreen1 AmCyan1

5 CherryPicker Cell Labeling & Capture CherryPicker systems provide a powerful sample preparation tool for monitoring and capturing a homogeneous population of cells that either: A) exhibit a specific promoter activity, or B) express a specific protein of interest Benefits of CherryPicker A universal, low-background magnetic bead-based cell capture system. Ideal for expression profiling, marker discovery, and posttranslational modification applications. Gentle enrichment of a homogeneous cell population under sterile conditions (unlike FACS). Simple and convenient bench-top protocol. How does CherryPicker work? The system utilizes a chimeric membrane-anchored fluorescent protein, CherryPicker, which can be monitored via fluorescence microscopy, and captured on magnetic beads via a specific antibody. CherryPicker is only expressed under the control of your promoter of interest, or together with your protein of interest via a bicistronic IRES expression cassette (Figure 3). Therefore, only the cells with your activity of interest will be enriched (Figure 4). A P CMV Enrich cells expressing your protein of interest Your gene IRES CherryPicker gene CherryPicker protein CherryPicker antibody Your protein kd Untransfected 35 x 103 cells 52.5 x 103 cells Before capture 35 x 103 cells 17.5 x 103 cells After capture Phospho-p44/42 MAPK (Erk 1/2) Magnetic beads B Enrich cells with your active promoter GAPDH TF CherryPicker gene Your promoter Expression Binding Capture Figure 3. CherryPicker System. Panel A. Insert your gene of interest into the IRES reporter vector. Your protein will be expressed with CherryPicker. Panel B. Insert your promoter of interest into the promoterless reporter vector. CherryPicker will be expressed under the control of your promoter. CherryPicker is targeted to the membrane, where it can be visualized by microscopy, and/or captured via magnetic beads. Figure 4. Enrichment of MEK-CA-expressing cells via CherryPicker Systems. HEK-293 cells were transiently transfected with the IRES-CherryPicker expression vector encoding a constitutively active mutant of MEK (MEK-CA). Transfected cells were mixed 1:10 with untransfected cells. The mixed (Lanes 2, 3 & 4) and untransfected (Lane 1) cell lysates were analyzed by Western blot before and after capture. Western blot analysis was performed using a phosphospecific antibody against phosphorylated ERK1/2. GAPDH served as a loading control for total protein. Cat. # Product Package Size CherryPicker Assay Kit 60 rxns 120 rxns CherryPicker Cell Capture System 60 rxns CherryPicker Cell Capture System (IRES) 60 rxns Lenti-X CherryPicker Cell Capture System 60 rxns Lenti-X CherryPicker Cell Capture System (IRES) 60 rxns

6 Visit for a complete vector list. Cat. # Product Package Size Bacterial Expression pacgfp1 Vector 20 μg pamcyan Vector 20 μg pdsred-express Vector 20 μg pdsred-express2 Vector 20 μg pe2-crimson Vector 20 μg Subcellular Labeling ppamcherry-mito Vector 10 μg pacgfp1-tubulin Vector 20 μg pautophagsense Vector 20 μg pdsred2-er Vector 20 μg pacgfp1-golgi Vector 20 μg In Vivo Imaging ptd Tomato Vector 20 μg pmplum Vector 20 μg phcred1 Vector 20 μg pe2-crimson Vector 20 μg pzsgreen Vector 20 μg FRET pacgfp1-c1 Vector 20 μg pdsred-monomer-c1 Vector 20 μg Protein Dynamics & Half-Life pdendra2-n Vector 20 μg pdendra2-c Vector 20 μg ppamcherry-n1 Vector 10 μg ppamcherry-c1 Vector 10 μg Antibodies Living Colors DsRed Monoclonal 20 μl 200 μl Living Colors DsRed Polyclonal 100 μl Living Colors mcherry Monoclonal 100 μl Living Colors Anti-RCFP Polyclonal Pan 100 μl Living Colors A.v. Peptide 200 μl 1 ml Living Colors EGFP Monoclonal 100 μl Living Colors GFP Monoclonal 100 μl Living Colors Full-Length ZsGreen Polyclonal 100 μl Living Colors A.v. Monoclonal (JL-8) 20 μl 200 μl Living Colors Full-Length GFP Polyclonal 20 μl 100 μl Notice to Purchaser Your use of these products and technologies is subject to compliance with any applicable licensing requirements described on the product s web page at It is your responsibility to review, understand and adhere to any restrictions imposed by such statements. Takara Bio Europe SAS Clontech Laboratories [email protected] [email protected] Europe : +33.(0) Austria : Germany : Switzerland : United Kingdom : For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. Clontech, the Clontech logo, AutophagSENSE, CherryPicker, Lenti-X, and Living Colors are trademarks of Clontech Laboratories, Inc. All other marks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions Clontech Laboratories, Inc. This brochure is printed on 60% recycled paper. ZZXBR633398EU