For in vitro use only!
Shipping: shipped on blue ice
Storage Conditions: store components as indicated on data sheet
avoid freeze/thaw cycles
Shelf Life: 6 months
Cocktail of Cell Penetrating Peptides and proteins for internalization of nucleotides (CPP-C02S)
Atto488-dUTP (20 μl, 1 mM, MW 1092.2 Da, 1 μl = 1 μg) Sufficient for up to 100 transduction experiments (NU-803-488)
DMSO (25 ml) (CPP-A01S)
Bovine serum albumin (BSA, 1 g) (CPP-A02)
Chloroquine diphosphate salt (1 g); Harmful! (CPP-A03)
o-Phenanthroline (50 mg); Toxic! (CPP-A06S)
Concentrated acidic glycine buffer (10x, pH 3, 25 ml, dilute 1:10 with sterile water before use) (CPP-A07S)
For additionally needed apparates and materials please read general manual (2.2.1). Take into account equipment and assays to detect and observe internalized cargo e.g. by selective staining, fluorescence microscopy or cell lyses followed by PAGE-electrophoresis and/or blotting.
Dissolve the whole content of the vial JBS-NUcleoducin (CPP-C02S) in 250 μl sterile and oxygen free water according to the general manual. Use the solution immediately or store aliquots at -20 °C. Avoid freeze/thaw cycles. Please note that the cocktail has proteolytic activity and can form disulfides and S-oxide (Met) when stored in solution. Store the fluorescent cargo Atto-488-dUTP (NU-803-488) also at -20 °C and avoid frequent thawing and freezing. Store the other compounds at 4 °C. If stored correctly, Jena Bioscience guarantees a shelf life of 6 months.
The Kit allows internalization of nucleotides, nucleic acids and plasmid DNA as well as estimation of optimum conditions for cellular uptake of the fluorescence labelled cargo, Atto488-dUTP, into the cell line of your interest. Some components of the transduction cocktail contain a nuclear localization sequence and are therefore able to transport a cargo into the nucleus.
The Kit further contains compounds for increasing rate and efficiency of transduction. DMSO enhances the permeability of cell membranes. BSA protects to some degree the peptide components of the cocktail against enzymatic degradation and stimulates simultaneously the uptake. Cells with secreted or membrane bound high proteolytic activity require the use of the protease inhibitor o-phenanthroline. Chloroquine triggers the release of cargo from intracellular vesicles. The complex which is only bound to the outer site of the cell membrane can be removed by repeated washing with acidic glycine buffer.
The procedures and their backgrounds are more detailed described in the general manual.
Handbook of Cell-Penetrating Peptides, Second Edition, Ed. by Ü. Langel, CRC Taylor and Francis, Boca Raton, London, New York (2007).
Morris et al. (2008) Cell penetrating peptides: from molecular mechanisms to therapeutics. Bio.Cell 100:201.
Morris et al. (2001) A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotechnol. 19:1173.
Covic et al. (2002) Activation and inhibition of G protein-coupled receptors by cell-penetrating membrane-tethered peptides. Proc Natl. Acad. Sci. 99:643.
Gros et al. (2006) A non-covalent peptide-based strategy for protein and peptide nucleic acid transduction. Biochim. Biophys. Acta 1758:384.
Snyder et al. (2005) Recent advances in the use of protein transduction domains for the delivery of peptides, proteins and nucleic acids in vivo. Expert Opin. Drug Deliv. 2:43.
Deshayes et al. (2008) Delivery of proteins and nucleic acids using a non-covalent peptide-based strategy. Adv. Drug Deliv. Rev. 60:537.
Availability Restriction: Exclusively distributed in Japan by Greiner BioOne