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17-10155 LentiBrite™ Paxillin-RFP Lentiviral Biosensor

MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
Key Applications: Transfection, Immunofluorescence, Immunocytochemistry
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17-10155
1 vial (minimum of 3 x 10E8 IFU/mL)  
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      Overview

      Replacement Information

      Key Specifications Table

      Key ApplicationsDetection Methods
      TFX, IF, ICCFluorescent
      Description
      Catalogue Number17-10155
      Trade Name
      • LentiBrite
      • Chemicon
      DescriptionLentiBrite™ Paxillin-RFP Lentiviral Biosensor
      OverviewRead our application note in Nature Methods!
      http://www.nature.com/app_notes/nmeth/2012/121007/pdf/an8620.pdf
      (Click Here!)

      Learn more about the advantages of our LentiBrite Lentiviral Biosensors! Click Here

      Biosensors can be used to detect the presence/absence of a particular protein as well as the subcellular location of that protein within the live state of a cell. Fluorescent tags are often desired as a means to visualize the protein of interest within a cell by either fluorescent microscopy or time-lapse video capture. Visualizing live cells without disruption allows researchers to observe cellular conditions in real time.

      Lentiviral vector systems are a popular research tool used to introduce gene products into cells. Lentiviral transfection has advantages over non-viral methods such as chemical-based transfection including higher-efficiency transfection of dividing and non-dividing cells, long-term stable expression of the transgene, and low immunogenicity.

      EMD Millipore is introducing LentiBrite™ Lentiviral Biosensors, a new suite of pre-packaged lentiviral particles encoding important and foundational proteins of autophagy, apoptosis, and cell structure for visualization under different cell/disease states in live cell and in vitro analysis.
      • Pre-packaged, fluorescently-tagged with GFP & RFP
      • Higher efficiency transfection as compared to traditional chemical-based and other non-viral-based transfection methods
      • Ability to transfect dividing, non-dividing, and difficult-to-transfect cell types, such as primary cells or stem cells
      • Non-disruptive towards cellular function

      EMD Millipore’s LentiBrite™ Paxillin-RFP lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of focal adhesion dynamics in difficult-to-transfect cell types.
      Background InformationAdherent cells maintain contact with the extracellular substratum by means of focal adhesions, comprising transmembrane matrix receptors (integrins) linked on their cytoplasmic side to a complex of signaling and structural proteins, which are in turn linked to the cytoskeleton. Paxillin, one of the first proteins recruited to nascent adhesions, serves as a scaffolding protein for kinases (Src, FAK), GTP exchange factors and other structural proteins, and plays a key role in assembly and disassembly of focal adhesions. Focal adhesions are highly dynamic structures that assemble at the leading edge of the migrating cell and disassemble at the trailing edge, and fusions between paxillin and fluorescent proteins have been widely employed for analysis of focal adhesion dynamics in live cells.
      EMD Millipore’s LentiBrite™ Paxillin-RFP lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of focal adhesion dynamics in difficult-to-transfect cell types.
      References
      Product Information
      Components
      • Paxillin-TagRFP Lentivirus:
        One vial containing 25 µL of lentiviral particles at a minimum of 3 x 10E8 infectious units (IFU) per mL.
        For lot-specific titer information, please see “Viral Titer” in the product box above.
      • Promoter
        EF-1 (Elongation Factor-1)
      • Multiplicty of Infection (MOI)
        MOI = Ratio of # of infectious lentiviral particles (IFU) to # of cells being infected.
        Typical MOI values for high transduction efficiency and signal intensity are in the range of 20-40. For this target, some cell types may require lower MOIs (e.g., HT-1080, human mesenchymal stem cells (HuMSC)), while others may require higher MOIs (e.g., human umbilical vein endothelial cells (HUVEC), U2OS, HeLa).
        NOTE: MOI should be titrated and optimized by the end user for each cell type and lentiviral target to achieve desired transduction efficiency and signal intensity.
      Detection methodFluorescent
      Quality LevelMQ100
      Applications
      Key Applications
      • Transfection
      • Immunofluorescence
      • Immunocytochemistry
      Application NotesFluorescence microscopy imaging:
      (See Figure 1 in datasheet)
      REF52 cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 40 for 24 hours. After media replacement and 48 hours further incubation, cells were fixed with formaldehyde and mounted. Images were obtained by oil immersion wide-field fluorescence microscopy. The paxillin-RFP displays a punctuate distribution at focal adhesions in cells.

      Immunocytochemistry comparison and inhibitor analysis:
      (See Figure 2 in datasheet)
      Similar to Figure 1 (see datasheet), HeLa cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 5 for 24 hours. After 24 hours, media was replaced and cells were then further incubated for 48 hours. Immunocytochemical staining (green) of the same fields of view with a monoclonal antibody against paxillin (Cat No. 05-417) reveals similar expression patterns to the protein-RFP (red).

      Additional cell types:
      (See Figure 3 in datasheet)
      U2OS and HT1080 cells were treated as in Figure 1 (see datasheet) at an MOI of 5 and 10, respectively, for 24 hours.

      Hard-to-transfect cell types:
      (See Figure 4 in datasheet)
      Primary cell type HUVEC and Human mesenchymal stem cells (HuMSC) were plated in chamber slides and transduced with lentiviral particles at an MOI of 5 and 20, repectively, for 24 hours.

      For optimal fluorescent visualization, it is recommended to analyze the target expression level within 24-48 hrs after transfection/infection for optimal live cell analysis, as fluorescent intensity may dim over time, especially in difficult-to-transfect cell lines. Infected cells may be frozen down after successful transfection/infection and thawed in culture to retain positive fluorescent expression beyond 24-48 hrs. Length and intensity of fluorescent expression varies between cell lines. Higher MOIs may be required for difficult-to-transfect cell lines.
      Biological Information
      Gene Symbol
      • PXN
      Purification MethodPEG precipitation
      UniProt Number
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Quality AssuranceEvaluated by transduction of HT-1080 cells and fluorescent imaging performed for assessment of target localization and transduction efficiency.
      Usage Statement
      • This product contains genetically modified organisms (GMO). Within the EU GMOs are regulated by Directives 2001/18/EC and 2009/41/EC of the European Parliament and of the Council and their national implementation in the member States respectively. This legislation obliges MilliporeSigma to request certain information about you and the establishment where the GMOs are being handled. Click here for Enduser Declaration (EUD) Form.
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
      Storage and Shipping Information
      Storage ConditionsStorage and Handling
      Lentivirus is stable for at least 4 months from date of receipt when stored at -80°C. After first thaw, place immediately on ice and freeze in working aliquots at -80°C. Frozen aliquots may be stored for at least 2 months. Further freeze/thaws may result in decreased virus titer and transduction efficiency.

      IMPORTANT SAFETY NOTE
      Replication-defective lentiviral vectors, such as the 3rd Generation vector provided in this product, are not known to cause any diseases in humans or animals. However, lentiviruses can integrate into the host cell genome and thus pose some risk of insertional mutagenesis. Material is a Risk Group 2 and should be handled under BSL2 controls. A detailed discussion of biosafety of lentiviral vectors is provided in Pauwels, K. et al. (2009). State-of-the-art lentiviral vectors for research use: Risk assessment and biosafety recommendations. Curr. Gene Ther. 9: 459-474.
      Packaging Information
      Material Size1 vial (minimum of 3 x 10E8 IFU/mL)
      Transport Information
      Supplemental Information
      Specifications
      Global Trade Item Number
      Catalog Number GTIN
      17-10155 04053252549212

      Documentation

      LentiBrite™ Paxillin-RFP Lentiviral Biosensor SDS

      Title

      Safety Data Sheet (SDS) 

      LentiBrite™ Paxillin-RFP Lentiviral Biosensor Certificates of Analysis

      TitleLot Number
      LentiBrite Paxillin-RFP Lentiviral Biosensor - 2201534 2201534
      LentiBrite™ Paxillin-RFP 2803283
      LentiBrite™ Paxillin-RFP -2572070 2572070
      LentiBrite™ Paxillin-RFP Lentiviral Biosensor 3073833
      LentiBrite™ Paxillin-RFP Lentiviral Biosensor - 3261281 3261281
      LentiBrite™ Paxillin-RFP Lentiviral Biosensor - 3448842 3448842
      LentiBrite™ Paxillin-RFP Lentiviral Biosensor - 3918062 3918062
      LentiBrite™ Paxillin-RFP Lentiviral Biosensor - 4046252 4046252
      LentiBrite™ Paxillin-RFP Lentiviral Biosensor Packaged Lentiviral Particles 3010025
      LentiBrite™ Paxillin-RFP Lentiviral Biosensor Packaged Lentiviral Particles 2943147