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Transduction Kit for Nucleoside Triphosphates

Cell Penetration

Cat. No. Amount Price (EUR) Buy / Note
CPP-K02 1 Kit (10 to 100 transductions) 300,00 Add to Basket/Quote Add to Notepad

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

Description:
Kit Components:

JBS-NUcleoducin
Cocktail of Cell Penetrating Peptides and proteins for internalization of nucleotides (CPP-C02S)

Fluorescent cargo
Atto488-dUTP (20 μl, 1 mM, MW 1092.2 Da, 1 μl = 1 μg) Sufficient for up to 100 transduction experiments (NU-803-488)

Auxiliary Compounds
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)

Wash Buffer
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.

Storage:
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.

Applications:
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.

Instructions:
The procedures and their backgrounds are more detailed described in the general manual.

  • Cultivate your cells.
  • Wash accordingly to the general manual.
  • Adjust amount of stock solution accordingly to the general manual (2.2.3). 3 μl of stock solution are able to form a non-covalent complex with 1 μg (1 μl) Atto488-dUTP or with 1 μg of an unlabelled nucleotide with a MW of 500 Da and 4 positive charges.
  • Calculate needed volumes of buffers, serum-free medium and serum with FCS according to the final volume.
  • Perform complex formation and transduction accordingly to the general manual (2.2.3, 2.2.4).
  • Add DMSO (10 %), BSA (0.5 to 1 %) and protease inhibitor o-phenanthroline (1 mM, MW = 198.2 Da) to the serum-free medium to increase rate and efficiency of transduction.
  • If microscopically indicated add chloroquine (150 μM, MW = 515 Da) to the serum-containing medium to release internalized cargo from vesicles.
  • Prolong serum-free transduction time from 1 to 4 h if necessary
  • After finishing transduction wash the cells thoroughly, use acidic glycine buffer.
  • Check the cells either with a fluorescence microscope or by FACS-analysis or by SDS-PAGE-electrophoresis.

Selected References:
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