Protein Delivery Protocols  

PROFECT PROTOCOL

 

The protocol is provided as an example for a 6-well plate format. Amounts for other plate sizes are given in Table 1.

 

Vortex the Profect P-1 reagent at full speed for 30 seconds once, just before use. Store the Profect-P-1 reagent at –20 oC. The Profect-P2 reagent can be stored at 4 o C or at -20 oC. 

 

Protocol:

 

  1. Set up cells to be transfected in Labtek-chamber slides so that they are about 80% confluent at the time of the experiment.
  2. Add 0.5-5 µl of protein solution (100 ng to 10 µg, in general we recommend 5 µg) to a sterile tube containing the appropriate amount of serum-free DMEM
  3. Add 3 µl or 5 µl of Profect reagent (mix well before use)
  4. Gently mix the transfection complex mixture by flicking the tube.
  5. Incubate at room temperature for 20 minutes
  6. Remove serum-containing growth media from cells by aspirating, wash cells with serum-free medium and add 1 ml of serum-free medium to each well.
  7. Add the transfection complex mixture to cells
  8. Return plate to incubator and incubate for 2-5 hours.
  9. Add 1 ml of complete media (containing 10% serum) to each well.
  10. Replace media on the following day and continue incubation until assaying. Wash cells with serum-free medium before assaying to remove any untransfected protein.

 

 

 

 

 

 

* The transfection complex mixture is composed of protein and Profect Transfection Reagent in serum-free medium. For example, at the incubation step (step 8) (6-well format), transfection complex mixture consisting of 2 µg protein, and 3 µl Profect Transfection Reagent in 200 μl serum-free medium is added to a well containing cells in a 1 ml volume.
 

Table 1: Protein transfection in different plat formats

Culture

Vessel

Volume of

Plating Medium

(per well)

Protein (µg)

In serum-free medium

Profect transfection complex mixture*

96 well

100 µl

0.1-0.2 µg in 10 µl

0.2 µl

24 well

200 µl

0.2-1.0 µg in 50 µl

0.2-2 µl

12 well

0.5 ml

0.5-2.0 µg in 100 µl

0.4-4 µl

35 mm dish

1 ml

0.5-2.0 µg in 100 µl

1-10 µl

6 well

1 ml

0.5-2.0 µg in 100 µl

1-10 µl

60 mm dish

2 ml

2.0-12 µg in 0.5 ml

4-24 µl

10 cm dish

7 ml

6.0-30 µg in 1.0 ml

6-60 µl

 

  

 

Peptide Delivery:

 

Suggested protocol for peptide delivery:

 

Mix 6.5 µg peptide with 5 µl of the P-2 reagent in 100 µl of high glucose DMEM.  Mix well and incubate at room temperature for 20 minutes then Vortex for 15 seconds. Dilute the complexes to 1 ml with high glucose DMEM and follow the transfection protocols recommended above.

 

For 96 well plates: Mix well and add 40 µl of complex per well of a 96 well plate (aspirate culture media before addition of transfection complex.)  Incubate at 37 °C for 3 hrs.  Add 100 µl of complete media and continue incubation.  Wash cells 4 times with serum free media and assay. 

 

 

TROUBLESHOOTING:

Toxicity may be observed when using low cell densities, or very small amounts of protein.  Usually an excess of uncomplexed Profect reagent may show some toxicity so if toxicity is observed when transfecting with very small amounts of protein (less than 100 ng) we recommend adding a carrier protein (e.g. β-galactosidase) so that the final protein concentration is about 2 µg total protein complexed with 5 µl of Profect.  Another alternative is to use lesser amounts of Profect for complex formation or to dilute the transfection complexes by 40%.  Increase the cell density, increase the amount of protein used for complex formation.  We strongly recommend contacting our tech support by email targsys@aol.com

 

Note:  In all instances the transfection complexes should be diluted at least 3X with complete media at the end of the recommended incubation periods for different proteins. In case of longer incubation periods please ensure that the cells are well covered with the protein-Profect mixes.

 

Protein of interest                    Suggested incubation period

 

Enzymes                                               2 hours

                                                                                   

Antibodies                                             5 hours and 24 hours

 

Histone                                                 3 hours and 12 hours

 

Low molecular weight proteins.            2-3 hours

 

Peptides                                               3 hours

 

After the suggested incubation periods, you can wash off the transfection complexes by washing cells extensively (4 times with DMEM and assessing protein delivery without fixing the cells). Alternatively, you can aspirate the transfection complexes at the end of the suggested incubation period and add complete media and wait longer to assess effects of protein delivery on the cells. NOTE: We recommend using Serum-Free, high glucose DMEM in place of OptiMem 1 as we have observed that it increases cell survival. DMEM can also be used as complexing medium for other applications.

REFERENCES CITING USE OF PROFECT REAGENTS:

 

  1. Nandan D, Yi T, Lopez M, Lai C, Reiner NE. (2002) Leishmania EF-1alpha activates the Src homology 2 domain containing tyrosine phosphatase SHP-1 leading to macrophage deactivation. J. Biol Chem. 2002 Dec 20;277(51):50190-7. Murine Macrophage cells

 

  1. Miao EA, Alouche-Aranda CM, Dors M, Clark AE, Bader MW, Miller SI amd Aderem A (2006) Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1 via Ipaf.  Nature Immunology,  7 , 569-575

 

  1. Genes to Cells 8 (12); 995-1003 Human Epidermoid Carcinoma cells

 

  1. Sendide K, Deghmane A’E, Pechkovsky D, Av-Gay Y, Mycobacterium bovis BCG Attenuates Surface Expression of Mature Class II Molecules through IL-10-Dependent Inhibition of Cathepsin S.  Khalid Sendide, Ala-Eddine Deghmane, Dmitri Pechkovsky, Yossef Av-Gay, Amina Talal, and Zakaria Hmama
    J. Immunol., Oct 2005; 175: 5324 - 5332.

 

  1. Nandan D, Yi T, Lopez M, Lai C, Reiner NE. (2002) Leishmania EF-1alpha activates the Src homology 2 domain containing tyrosine phosphatase SHP-1 leading to macrophage deactivation.  J Biol Chem. 2002 Dec 20;277(51):50190-7.

 

  1. Sato, Ken-ichi, Nagao, Tomomi, Iwasaki, Tetsushi, Nishihira, Yusuke & Fukami, Yasuo (2003) Src-dependent phosphorylation of the EGF receptor Tyr-845 mediates Stat-p21waf1 pathway in A431 cells.  Genes to Cells 8 (12), 995-1003. 3. Miao EA, Alouche-Aranda CM, Dors M, Clark AE, Bader MW, Miller SI amd Aderem A

 

  1. T. Takami, S. Terai, Y. Yokoyama, H. Tanimoto, K. Tajima, K. Uchida, T. Yamasaki, I. Sakaida, H. Nishina, S. Thorgeirsson Human Homologue of Maid Is a Useful Marker Protein in Hepatocarcinogenesis. Gastroenterology, Volume 128, Issue 5, Pages 1369-1380

 

  1. Hirokazu Tanaka, Itaru Matsumura, Kiminari Itoh, Asako Hatsuyama, Masayuki Shikamura, Yusuke Satoh, Toshio Heike, Tatsutoshi Nakahata, Yuzuru Kanakura (2007) HOX Decoy Peptide Enhances the Ex Vivo Expansion of Human Umbilical Cord Blood CD34+ Hematopoietic Stem Cells/Hematopoietic Progenitor Cells.  Stem Cells Vol. 24 No. 11 November 2006, pp. 2592 -2602

 

  1. Hafid Soualhine, Ala-Eddine Deghmane, Jim Sun, Karen Mak, Amina Talal, Yossef Av-Gay, and Zakaria Hmama Hafid Soualhine, Ala-Eddine Deghmane, Jim Sun, Karen Mak, Amina Talal, Yossef Av-Gay, and Zakaria Hmama. Mycobacterium bovis Bacillus Calmette-Guérin Secreting Active Cathepsin S Stimulates Expression of Mature MHC Class II Molecules and Antigen Presentation in Human Macrophages J. Immunol., Oct 2007; 179: 5137 – 5145

 

  1. D Dalkara, G Zuber, and JP Behr Intracytoplasmic delivery of anionic proteins. Mol Ther, Jun 2004; 9(6): 964-9.