Выделение и характеристика химерных человека Fc-экспрессии белков с использованием белок мембраны адсорберов и рационализировать рабочий процесс

1Department of Chemical and Biomolecular Engineering, Ohio University, 2Biomedical Engineering Program, Russ College of Engineering and Technology, Ohio University, 3Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School
Bioengineering

Your institution must subscribe to JoVE's Bioengineering section to access this content.

Fill out the form below to receive a free trial or learn more about access:

Welcome!

Enter your email below to get your free 10 minute trial to JoVE!





We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.

If you want more info regarding data storage, please contact gdpr@jove.com.

 

Cite this Article

Copy Citation | Download Citations

Burdick, M. M., Reynolds, N. M., Martin, E. W., Hawes, J. V., Carlson, G. E., Cuckler, C. M., Bates, M. C., Barthel, S. R., Dimitroff, C. J. Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow. J. Vis. Exp. (83), e51023, doi:10.3791/51023 (2014).

Please note that all translations are automatically generated.

Click here for the english version. For other languages click here.

Abstract

Materials

Name Company Catalog Number Comments
Protein A membrane adsorber (Sartobind Protein A 2 ml)  Sartorius-Stedim 93PRAP06HB-12--A
Amicon Ultra centrifugal filter, 10 kDa, 4 ml Millipore UFC801008 Use 15 ml volume if needed
Sterile vacuum filter unit, 0.22 µm, 500 ml Millipore SCGPU05RE
10 ml Syringes with Luer Lock tips BD 301604
30 ml Syringes with Luer Lock tips BD 309650
Tygon lab tubing (1/16 in x 1/8 in x 50 ft) Cole Parmer WU-95903-16
Slide-A-Lyzer MINI Dialysis Devices (10K MWCO) Thermo Scientific 69576
Snap i.d. antibody collection tray EMD Millipore WBAVDABTR
Snap i.d. single well blot holder EMD Millipore WBAVDBH01
Elution buffer Thermo Scientific 21004
Tris, ultra pure Fisher Scientific 819623
Sodium chloride Fisher Scientific 7647-14-5
Tween 20 Fisher Scientific BP337-100
DPBS Thermo Scientific SH30028.02
DPBS with Ca2+/Mg2+ Life Technologies 14080-055
BSA Sigma A9647
Human Fc Bethyl Research Laboratories P80-104
Anti-human Fc-APC Jackson Immunoresearch 109136170
Anti-human Fc-AP Bio-Rad 170-5018
Alkaline phosphatase substrate Promega S3841
Flow cytometry tubes (5 ml polystyrene or polypropylene) BD 352054
Syringe pump Harvard Apparatus 55-2226 A peristaltic pump is also acceptable
Protein gel electrophoresis system Bio-Rad Laboratories 552BR094876 Any gel electrophoresis system is acceptable, but mini gel systems run fastest
Semidry blotter Bio-Rad Laboratories 690BR006163 Any transfer system is acceptable, but discontinuos transfer systems perform electrophoretic transfer fastest
SNAP i.d. vacuum-assisted protein detection system EMD Millipore WBAVDBASE An upgraded model has replaced this specific instrument, but either should work just as well

DOWNLOAD MATERIALS LIST

References

  1. Gagnon, P. Technology trends in antibody purification. J. Chromatogr. A. 1221, 57-70 (2012).
  2. Gottschalk, U. Bioseparation in antibody manufacturing: The good, the bad and the ugly. Biotechnol. Prog. 24, 496-503 (2008).
  3. Liu, H. F., Ma, J., Winter, C., Bayer, R. Recovery and purification process development for monoclonal antibody production. MAbs. 2, 480-499 (2010).
  4. Low, D., O'Leary, R., Pujar, N. S. Future of antibody purification. J. Chromatogr. B Anal. Technol. Biomed. Life. Sci. 848, 48-63 (2007).
  5. Boi, C. Membrane adsorbers as purification tools for monoclonal antibody purification. J. Chromatogr. B Anal. Technol. Biomed. Life. Sci. 848, (1016).
  6. Cuatrecasas, P. Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J. Biol. Chem. 245, 3059-3065 (1970).
  7. Shukla, A. A., Gottschalk, U. Single-use disposable technologies for biopharmaceutical manufacturing. Trends Biotechnol. 31, 147-154 (2013).
  8. Boi, C., Dimartino, S., Sarti, G. C. Performance of a new protein a affinity membrane for the primary recovery of antibodies. Biotechnol Prog. 24, 640-647 (2008).
  9. Dancette, O. P., Taboureau, J., Tournier, E., Charcosset, C., Blond, P. Purification of immunoglobulins g by protein a/g afinity membrane chromatography. J. Chromatogr. B. 723, 61-68 (1999).
  10. Thommes, J., Etzel, M. Alternatives to chromatographic separations. Biotechnol. Prog. 23, 42-45 (2007).
  11. Boi, C., Dimartino, S., Sarti, G. C. Modelling and simulation of affinity membrane adsorption. J. Chromatogr. A. 1162, 24-33 (2007).
  12. van Beijeren, P., et al. Computer-aided process design of affinity membrane adsorbers: A case study on antibodies capturing. Chem. Pap. 62, 458-463 (2008).
  13. Lauriere, M. A semidry electroblotting system efficiently transfers both high- and low-molecular-weight proteins separated by sds-page. Anal. Biochem. 212, 206-211 (1993).
  14. Darcy, E., Leonard, P., Fitzgerald, J., Danaher, M., O'Kennedy, R. Purification of antibodies using affinity chromatography. Methods Mol. Biol. 681, 369-382 (2011).
  15. Hjelm, H., Hjelm, K., Sjoquist, J. Protein a from staphylococcus aureus. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins. FEBS Lett. 28, 73-76 (1972).
  16. Hober, S., Nord, K., Linhult, M. Protein a chromatography for antibody purification. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 848, 40-47 (2007).
  17. Kronvall, G. A surface component in group a, c, and g streptococci with non-immune reactivity for immunoglobulin g. J. Immunol. 111, 1401-1406 (1973).
  18. Cedeno-Laurent, F., et al. Development of a nascent galectin-1 chimeric molecule for studying the role of leukocyte galectin-1 ligands and immune disease modulation. J. Immunol. 185, 4659-4672 (2010).
  19. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage t4. Nature. 227, 680-685 (1970).
  20. Towbin, H., Staehelin, T., Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354 (1979).
  21. Reisner, A. H., Nemes, P., Bucholtz, C. The use of Coomassie brilliant blue g250 perchloric acid solution for staining in electrophoresis and isoelectric focusing on polyacrylamide gels. Anal. Biochem. 64, 509-516 (1975).
  22. Cedeno-Laurent, F., et al. Metabolic inhibition of galectin-1-binding carbohydrates accentuates antitumor immunity. J. Invest. Dermatol. 132, 410-420 (2012).
  23. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 (1976).
  24. Desjardins, P., Hansen, J. B., Allen, M. Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer. J. Vis. Exp. (33), (2009).
  25. Ernst, O., Zor, T. Linearization of the Bradford Protein Assay. J. Vis. Exp. (38), (2010).
  26. Simonian, M. H., Smith, J. A. Spectrophotometric and colorimetric determination of protein concentration. Curr. Protoc. Mol. Biol. Chapter. 10, 10-1002 (2006).
  27. Smith, P. K., et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85 (1985).
  28. Gravel, C., Li, C., Wang, J., Hashem, A. M., Jaentschke, B., Van Domselaar, G., et al. Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays. J. Vis. Exp. (50), (2011).

Comments

0 Comments


    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Usage Statistics