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Membrane proteins are major sensors of extracellular stimuli and initiators of intracellular signal transduction, and their abundance on the cell surface in particular is often dynamically regulated even when there are no significant changes of their total abundance in a cell. This protocol is designed to biochemically label and separate membrane proteins on the plasma membrane from those in the intracellular compartments. In conjunction with co-immunoprecipitation and western blot analysis, functional analysis of dynamic interaction of membrane proteins with other membrane proteins or intracellular adaptor and effector proteins can be achieved.

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Biotinylation of Cell Surface Proteins

Biochemistry > Protein > Modification
Author: Guo N. Huang
Guo N. HuangAffiliation: Program in Biochemistry, Cellular and Molecular Biology, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: guo.n.huang@gmail.com
Bio-protocol author page: a30
Vol 2, Iss 9, 5/5/2012, 16221 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.170

[Abstract] Membrane proteins are major sensors of extracellular stimuli and initiators of intracellular signal transduction, and their abundance on the cell surface in particular is often dynamically regulated even when there are no significant changes of their total abundance in a cell. This protocol is designed to biochemically label and separate membrane proteins on the plasma membrane from those in the intracellular compartments. In conjunction with co-immunoprecipitation and western blot analysis, functional analysis of dynamic interaction of membrane proteins with other membrane proteins or intracellular adaptor and effector proteins can be achieved.

Materials and Reagents

  1. HEK293 cells
  2. Poly-dL-Ornithine (PLO) (Sigma-Aldrich, catalog number: P8638)
  3. Boric Acid (Sigma-Aldrich, catalog number: B0394)
  4. Sulfo-NHS-SS-biotin solution (Thermo Fisher Scientific, Pierce Antibodies, catalog number: 21331)
  5. Neuroavidin beads (Thermo Fisher Scientific, Pierce Antibodies, catalog number: 29200)
  6. EDTA•2H2O (MW 372.2) (Sigma-Aldrich, catalog number: E1644)
  7. EGTA•2H2O (MW 380.4) (Sigma-Aldrich, catalog number: E4378)
  8. NaPyrophophate (Sigma-Aldrich, catalog number: S9515)
  9. NaF (Sigma-Aldrich, catalog number: S7920)
  10. Na3VO4•10H2O (MW 446.1) (Sigma-Aldrich, catalog number: 5-9515)
  11. Protease inhibitor (F. Hoffmann-La Roche, catalog number: 1873580)
  12. NaVO3
  13. NaOH
  14. PBS
  15. CaCl2
  16. MgCl2
  17. Glycine
  18. Triton
  19. HCl
  20. 4x loading buffer
  21. Biotin quenching solution (see Recipes)
  22. PBS/CaCl2/MgCl2 (see Recipes)
  23. IP buffer (see Recipes)
  24. Stock (see Recipes)

Equipment

  1. 0.22 μm filters
  2. 12-well plates
  3. Western blot equipment

Procedure

  1. Prepare PLO solution: Add 90 mg PLO into 180 ml borate buffer (dissolve 1.668 g borate acid in 180 ml H2O and adjust pH to 8.3 using NaOH), and sterilize by filtering through 0.22 μm filters.
  2. Coat 12-well plates with PLO solution at room temperature overnight in the culture hood (1 ml per well). Wash three times with sterile water on the second day before cell plating.
  3. Plate HEK293 cells on poly-ornithine coated 12-well plates and transfect as needed.
  4. On the day of cell harvest, dissolve Sulfo-NHS-SS-Biotin in PBS/CaCl2/MgCl2 at 0.5 mg ml-1 and store on ice.
  5. Wash cells with 1 ml ice-cold PBS/CaCl2/MgCl2 twice.
  6. Biotinylation
    1. Add 0.3 ml of 0.5 mg/ml Sulfo-NHS-SS-biotin solution.
    2. Incubate on ice for 30 min, with occasional shaking.
  7. Wash three times with ice-cold quenching solution and incubate 5 min before each solution removal to allow neutralization of uncrosslinked reagents.
  8. Lyse cells with 120 μl IP buffer and incubate 10 min on ice.
  9. Transfer all contents of each well into a 1.5-ml tube and spin at Vmax (about 14,000 rpm) on a 4 °C desktop centrifuge for 10 min.
  10. Save 10 μl supernatant (added 10 μl 4x loading buffer) as offers.
    Mix 100 μl supernatants with 50 μl 50% slurry of immobilized Neutravidin and rotate for 2-3 h at 4 °C.
  11. Spin 1 min at 5,000 rpm in a 4 °C centrifuge. Remove the supernatant and wash the beads with 1 ml IP buffer by rotating the mixture for 5 min in the 4 °C room. Wash three times in total.
  12. After the final wash, remove the supernatant and add 25 μl 4x loading buffer to the beads. Mix well and put the tubes in a 65 °C water bath for 10 min to denature proteins (65 °C is chosen because we find that in boiling water, many membrane proteins form aggregates, which results in failure of proteins to migrate into acrylamide gels). Load 10 μl for western blot analysis.

Recipes

  1. Biotin quenching solution (pH 7.4)
    50 mM glycine (0.1877 g) in 50 ml PBS/CaCl2/MgCl2
  2. PBS/CaCl2/MgCl2
    PBS (+2.5 mM CaCl2, 1 mM MgCl2, pH 7.4)
  3. IP buffer (pH 7.4)
    PBS

    50 ml
    EDTA
    5 mM
    0.5 ml x 0.5 M (pH 7.4)
    EGTA
    5 mM
    0.5 ml x 0.5 M (pH 7.4)
    NaPyrophophate
    10 mM
    0.223 g
    NaF
    50 mM
    0.1 g
    NaVO3   
    1 mM   
    0.5 ml x 100 mM
    1% Triton
    1 tablet Protease inhibitor
  4. Stock
    1. 0.5 M EDTA (pH 7.4)
      93.05 g EDTA•2H2O
      500 ml ddH2O
      Add NaOH until EDTA dissolves and add HCl to adjust pH back to 7.4.
    2. 0.5 M EGTA (pH 7.4)
      95.1 g EGTA•2H2O
      500 ml ddH2O
      Add NaOH until EDTA dissolves and add HCl to adjust pH back to 7.4.
    3. 100 mM Na3VO4
      1.84 g Na3VO4•10 H2O
      Add HCl to pH less than 10 (yellow solution). Boil the solution until clear and colorless.
    4. 10 ml aliquots and freeze at -20 °C.

Acknowledgments

This protocol was previously used in and adapted from Huang et al. (2006).

References

  1. Huang, G. N., Zeng, W., Kim, J. Y., Yuan, J. P., Han, L., Muallem, S. and Worley, P. F. (2006). STIM1 carboxyl-terminus activates native SOC, I(crac) and TRPC1 channels. Nat Cell Biol 8(9): 1003-1010.


How to cite this protocol: Huang, G. N. (2012). Biotinylation of Cell Surface Proteins. Bio-protocol 2(9): e170. DOI: 10.21769/BioProtoc.170; Full Text



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