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17-10154 LentiBrite™ Paxillin-GFP Lentiviral Biosensor

MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
Key Applications: Transfection, Immunofluorescence, Immunocytochemistry
17-10154
1 vial (minimum of 3 x 10E8 IFU/mL)  
Purchase on Sigma-Aldrich

Overview

Replacement Information

Key Spec Table

Key ApplicationsDetection Methods
TFX, IF, ICCFluorescent
Description
Catalogue Number17-10154
Trade Name
  • LentiBrite
  • Chemicon
DescriptionLentiBrite™ Paxillin-GFP 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-GFP lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of autophagy 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 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. Fusions between paxillin and fluorescent proteins have been widely employed for analysis of focal adhesion dynamics in live cells.
EMD Millipore’s LentiBrite™ Paxillin-GFP 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-TagGFP2 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, HeLa, U2OS, human mesenchymal stem cells (HuMSC)), while others may require higher MOIs (e.g., human umbilical vein endothelial cells (HUVEC)).
    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)
U2OS cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 5 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-GFP displays a punctate distribution at focal adhesions in cells.

Immunocytochemistry Comparison:
(See Figure 2 in datasheet)
Similar to Figure 1, HeLa cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 40 for 24 hours. After 24 hours, media was replaced and cells were then further incubated for 48 hours. Immunocytochemical staining (red) of the same fields of view with a monoclonal antibody against Paxillin (Cat No. 05-417) reveals similar expression patterns to the protein-GFP (green).

Hard-to-transfect Cell Types:
(See Figure 3 in datasheet)
Primary cell types HUVEC or HuMSC were plated in chamber slides and transduced with lentiviral particles at an MOI of 20 and 5, respectively, for 24 hours.

Additional Cell Types:
(See Figure 4 in datasheet)
HT1080 and REF52 cells were treated as in Figure 1.

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 at MOI values of 20 and 40. 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 Merck 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
Catalogue Number GTIN
17-10154 04053252640001

Documentation

LentiBrite™ Paxillin-GFP Lentiviral Biosensor SDS

Title

Safety Data Sheet (SDS) 

LentiBrite™ Paxillin-GFP Lentiviral Biosensor Certificates of Analysis

TitleLot Number
17-10154 - N1956533 N1956533
LentiBrite™ Paxillin-GFP -2603858 2603858
LentiBrite™ Paxillin-GFP -2817968 2817968
LentiBrite™ Paxillin-GFP Lentiviral Biosensor - 3160186 3160186
LentiBrite™ Paxillin-GFP Lentiviral Biosensor - 3288790 3288790
LentiBrite™ Paxillin-GFP Lentiviral Biosensor - 3715682 3715682
LentiBrite™ Paxillin-GFP Lentiviral Biosensor - 4045145 4045145
LentiBrite™ Paxillin-GFP Lentiviral Biosensor -2827680 2827680
LentiBrite™ Paxillin-GFP Lentiviral Biosensor -2886930 2886930
LentiBrite™ Paxillin-GFP Lentiviral Biosensor Packaged Lentiviral Particles 3093514
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Technical Info

Title
LentiBrite™ Lentiviral Biosensors for Fluorescent Cellular Imaging: Analysis of Autophagosome Formation