Paul.xu at 15:51, 19 June 2011

2011-06-19T15:51:17Z

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PEP-1 peptide with 21 amino acids (KETWWETWWTEWSQPKKKRKV), can be used to deliver full-length native proteins in vitro and in vivo.

Pep-1 peptide has three domains: (1) a hydrophobic tryptophan-rich motif containing five tryptophan residues (KETW WETWWTEW); (2) a hydrophilic lysine-rich domain (KKKRKV) derived from the nuclear localization sequence (NLS) of simian virus 40 (SV-40) large T antigen; (3) a spacer domain (SQP), separating the two domains mentioned above, containing a proline residue, which improves the flexibility and the integrity of both the hydrophobic and the hydrophilic domains.

The hydrophobic domain is required for efficiently targeting the cell membrane and for hydrophobic interactions with proteins

The hydrophilic domain is required to improve intracellular delivery

In standard cell culture conditions, Pep-1 localizes rapidly, in <10 min, to the nucleus of human HS-68, murine NIH-3T3 fibroblasts, or Cos cells. Similar experiments, performed by incubating cells for 30 min at 4°C before transfection, yielded essentially the same result, indicating that Pep-1 internalization is independent of normal endocytosis.

 

1. Seed 1 Ã 10^5 cells per well in 24-well dishes coated with poly-D-lysine. 2. Grow cells to 80% confluence in Neurobasal, B27, 20mM L-glutamine, 2% Fetal Bovine Serum containing 50 U/ml Interferon gamma (Peprotech) at 33°C with 6% CO2. 3. Replace medium with serum-free medium. 4. Dissolve 2 ÎŒl of Pep-1 stock suspension (Active Motif, Carlsbad, CA, USA) in 48 ÎŒl ddH2O. 5. Dilute 0.5-1 µg protein/antibody (or 100-500 ng peptide) in 50 ÎŒl PBS. 6. Add diluted protein to diluted Chariot and incubate at room temperature for 30 7. Incubate for 30 min at RT. 8. Add Chariot-macromolecule complex to cells 9. Incubate at 37ºC in a humidified atmosphere containing 5% CO2 for one hour.

Incubation time: Peptide: 0.5-1 hour

Protein: 1-2 hours

Antibody: 2 hour

10. Process the cells for observation or detection assay

'''For suspension culture cells''' 1. Collect the suspension cells by centrifugation at 200-400 x g for 5 minutes. Remove the supernatant.

2. Wash the cells twice with 1X PBS.

3. Centrifuge at 200-400 x g for 5 minutes to pellet the cells. Remove the supernatant.

4. Resuspend the cell pellet in the Chariot-macromolecule complex. Add serum-free medium to achieve the Final Transfection Volume

5. Incubate at 37ºC in a humidified atmosphere containing 5% CO2 for 1 hour to 3 hours
<tab border="1">
Plate Surface Area (mm^2 ) Cells Growth Medium (ml)
96-well 20 1.0 x 10^4 0.2
24-well 200 0.05 x 10^6 0.5-1.0
12-well 401 0.1 x 10^6 1-2
6-well or 35 mm 962 0.3 x 106 3-5
60 mm 2827 0.8 x 106 5
100 mm 7854 2.2 x 106 10
</tab>

<tab border="1">
Plate Sterile H20 for Chariot dilution (µl) Volume of Chariot (µl) Volume of protein in PBS(µl) Serum-Free Medium (µl) Final Transfection volumne (µl)
96-well 10 0.5 10 80 100
24-well 50 2 50 100 200
12-well 100 3.5 100 150 350
6-well or 35 mm 100 6 100 400 600
60 mm 200 20 1600 200 2000
100 mm 200 50 4600 200 6000
</tab>

Chariot concentration: 2 µg/µl Chariot is covered under U.S. Patent No. 6,841,535

[[Category:Protocols]]

Protein-Transfection - Revision history

Paul.xu at 15:51, 19 June 2011