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EntryPep Kit
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EntryPep™ KIT FOR PROTEIN DELIVERY
EntryPep™ Kit of Inbiolabs® provides a fast and simple method for delivering a protein of choice into a wide variety of cells via conjugation with a cell permeable polypeptide sequence (CP). It’s a convenient method for covalent modification of 0.2–2 mg of protein – for instance, an antibody – by a specific short polypeptide sequence. This cross-linking is based on the formation of a stable thioether bond. The resulting complex becomes cell membrane permeable but retains the biological activity of the modified protein (e.g. antigen recognition by antibody). This complex can be used in various assays such as visualization or functional modulation of intracellular proteins in living cells. The protocol is appropriate for all proteins containing thiol groups.

EntryPep™ Kit advantages 

  • The efficiency of internalization is much higher than with common DNA transfection methods.
  • The procedure is much faster than conventional transfection-based expression analysis.
  • The conjugate is equally distributed among all treated cells.
  • Functional effects can be measured immediately after internalization of the conjugate.
  • The protein level within the cell can be easily controlled and does not depend on the efficiency of transcription or translation.

The conjugated protein is internalized into the cell interior within 1-2 hours with high efficiency (penetrates all treated cells), and at a wide range of concentrations. Such a conjugate can be utilized in a variety of applications requiring the use of proteins that normally do not cross the cell membrane.

EntryPep™ Kit delivers:

  • Peptides
  • Proteins
  • Antibodies
  • Protein complexes
  • Chemically modified proteins
  • All other compounds with -SH group

EntryPep™ Kit applications

  • Analysis of subcellular distribution of dye-labelled proteins in living cells
  • Visualization of intracellular proteins with CP-conjugated antibodies or other specific ligands
  • Detection of co-localization of multiple CP-conjugated proteins in living cells
  • Verification of protein-protein interactions in a cellular environment (i.e. verification of results from two-hybrid assays)
  • Inhibition or activation of intracellular processes with a specific protein
  • Functional analysis of proteins that are difficult to express in mammalian cells (because of toxicity or low expression levels)
  • Rapid assessment of activity of recombinant proteins in a cellular environment
  • Evaluation of intracellular effects of chemically modified proteins (which cannot be done using DNA transfection based techniques)

Proven performance
EntryPep™Kit application exhibits: antibody internalization

Figure 1: Actin visualized with internalized Cy3-labelled anti-Actin antibody.
Figure 2: Endosomes visualized with internalized Alexa 488-labelled anti-Lamp1 antibody.

Contents and storage
EntryPep™ Kit contains enough reagents for 36 reactions.

Reagents and materials supplied:  
  1. Activated cell permeable sequence
  2. TCEP-HCl for use as positive control
  3. Spin columns, three 2 ml columns containing gel filtration resin
  4. Reaction buffer (phosphate buffered saline)

Storage: the kit should be stored at 4°C. Under these conditions, the kit components are stable for at least six months.

References

  • Pooga, M., et al, FASEB J. 1998 Dec: 67-77. Cell Penetration by transportan.
  • Pooga, M., et al, FASEB J. 2001;15:1451-1453. Cellular translocation of proteins by transportan.
  • Säälik, P., et al, Bioconjug Chem. 2004 Nov-Dec;15(6):1246-53. Protein cargo delivery properties of cell-penetrating peptides. A comparative study.
  • Hällbrink, M., et al, Biochim Biophys Acta. 2001 Dec 1;1515(2):101-9. Cargo delivery kinetics of cell-penetrating peptides.

 

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EntryPep Kit introduction