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This protocol can be used to purify membrane proteins from yeast samples under native conditions at a large scale. This protocol has been developed primarily for FLAG-tagged proteins. This protocol can however be slightly modified and applied to other tags, such as GST or HA.

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[Bio101] Large Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast

Biochemistry > Protein > Isolation and purification
Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicin, Baltimore , USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
1/5/2011, 10417 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.12

[Abstract] This protocol can be used to purify membrane proteins from yeast samples under native conditions at a large scale. This protocol has been developed primarily for FLAG-tagged proteins. This protocol can however be slightly modified and applied to other tags, such as GST or HA.

Materials and Reagents

  1. EDTA free protease inhibitors (Roche Diagnostics)
  2. Digitonin (EMD Chemicals)
  3. Protease Inhibitors (DMSO, leupeptin, pepstatin) (Sigma-Aldrich)
  4. ANTI-Flag M2 affinity gel (Sigma-Aldrich)
  5. 3x FLAG peptide (Sigma-Aldrich)
  6. NaF# (Ser/Thr phosphatase inhibitor)
  7. Na3VO4 (Tyr phosphatase inhibitor)
  8. HEPES
  9. KOAc
  10. Mg(OAc)2
  11. CaCl2
  12. Sorbitol
  13. Glycerol
  14. KOH
  15. PMSF
  16. DMSO
  17. Lysis buffer (see Recipes)
  18. Immunoprecipitation buffer (see Recipes)
  19. 50 ml lysis buffer (see Recipes) 
  20. 14 ml IP buffer with 2% digitonin (see Recipes)
  21. 20 ml IP buffer with 0.1% digitonin (see Recipes)
  22. 40 ml IP buffer with 0.1% digitonin (see Recipes)
  23. 3x FLAG elution buffer (see Recipes)

Equipment

  1. Avestin Emulsiflex C3 homogenizer (Avestin®)
  2. Beckman centrifuge and Type 70 Ti rotor (Beckman Coulter) 
  3. Beckman polycarbonate centrifuge tubes (Beckman Coulter, catalog number: 355631)
  4. 1 L centrifuge bottles
  5. 50 ml conical tubes

Procedure

  1. Preparation of cell lysate
    1. Collect 4,000 OD of cells collected at OD600~1.4. Split to 4x 1 L centrifuge bottles. Spin at 5,000 rpm for 20 min using Rotor JLA 8.100.
    2. Wash samples in each bottle with 100 ml H2O. Transfer to 50 ml conical tubes. Store at -80 °C if necessary.
    3. Resuspend cells in 50 ml ice-cold lysis buffer supplemented with protease inhibitors (no DTT) and phosphatase inhibitors (add 50 ml buffer to first bottle, vortex, transfer all to the next bottle and so on. Eventually you will have two 50 ml conical tubes of sample).
    4. Use Avestin Emulsiflex C3 homogenizer to disrupt cells.
    5. Clear unlysed cells by centrifugation at 1,000 x g twice or three times.
    6. Transfer supernatant to Beckman polycarbonate tubes (the filling level is 16.5 ml in Type 70 Ti rotor). Use a Type 70 Ti rotor for high-speed spin at 44,000 x g (24,453 rpm) for 30 min to pellet microsomes. 
    7. Microsomes are resuspended in 0.5 ml lysis buffer and then diluted with 14 ml immunoprecipitation buffer with 2% digitonin supplemented with protease inhibitors.
    8. Membrane proteins can be solubilized by nutating lysate at 4 °C for 1.5 h. Remove unsolubilized material by centrifugation at 44,000 x g for 30 min (24,453 rpm in a Type 70 Ti rotor).

  2. FLAG Fusion Protein Immunoprecipitation
    1. Use 300 μl of gel suspension per reaction (~150 μl of packed gel volume).
    2. Thoroughly suspend the ANTI-FLAG M2 affinity gel in the vial and transfer them to a 15 ml conical tube. 
    3. Centrifuge the resin for 1 min at 400 x g. Wait for 1-2 min before handling the samples. Remove the supernatant with a gel-loading pipette tip.
    4. Wash the packed gel 4x with 5 ml of immunoprecipitation buffer with 0.1% digitonin. 
    5. Add the ~15ml cell lysate to the ANTI-FLAG resin. Immuoprecipitations are rotated for 3 h or overnight at 4 °C.
    6. Wash the resin 4x with 10 ml immunoprecipitation buffer with 0.1% digitonin. Transfer the beads into an Eppendorf tube.

  3. Elution of the FLAG –fusion protein
    Elution with 3x FLAG peptide. This elution efficiency is very high using this method.
    1. Prepare 3x FLAG Elution Buffer. Add 150 μl of 1x immunoprecipitation buffer with 0.25% digitonin and 1 μg/μl 3x FLAG peptide.
    2. Add 150 μl of 3x FLAG elution buffer to the resin in the test tube.
    3. Incubate the samples with gentle shaking for 30 min at 4 °C.
    4. Centrifuge the resin for 1 min at 400~1000 x g. Transfer the supernatant to fresh test tubes. 
    5. Repeat eluation with another 150 μl of 3x FLAG elution buffer with 0.25% digitonin and 0.1 μg/μl 3x FLAG peptide. Combine the eluates.

Recipes

  1. Lysis buffer
    Lysis buffer
    1 L
    2x  buffer without detergent
    50 mM HEPES (FW 238.3)

    23.83 g
    50 mM KOAc* (FW 98.14)

    9.814 g
    2 mM Mg(OAc)2

    4 ml of 1 M Mg(OAc)2
    1 mM CaCl2

    20 ml of 0.1 M CaCl2
    200 mM sorbitol (FW 182.17)

    72.87 g
    1 mM NaF# (Ser/Thr phosphatase inhibitor)

    0.084 g
    0.3 mM Na3VO4 (Tyr phosphatase inhibitor)

    0.1103 g

  2. Immunoprecipitation buffer
    Immunoprecipitation buffer
    1 L
    2x buffer without detergent
    50 mM HEPES-KOH (pH 6.8)

    23.83 g
    50 mM KOAc* (FW 98.14)

    9.814 g
    2 mM Mg(OAc)2

    4 ml of 1 M Mg(OAc)2
    1 mM CaCl2

    20 ml of 0.1 M CaCl2
    15% glycerol (v/v)

    300 ml of 100% glycerol
    Add Mili-Q H2O to final volume, adjust to pH=6.8 using 0.5 M KOH.
    Add the following protease inhibitors per 50 ml immunoprecipitation buffer before use: 1 complete tablet, EDTA free protease inhibitors (crush tablet first in weigh paper).
    Add 100 μl of 0.5 M PMSF in DMSO.
  3. 50 ml lysis buffer
    25 ml 2x lysis buffer
    25 ml H2O
    1 EDTA free protease inhibitor tablet
    100 μl of 0.5 M PMSF in DMSO
  4. 14 ml of IP buffer with 2% digitonin
    7 ml of 2x IP buffer
    2.8 ml of 10% digitonin
    1ml EDTA free protease inhibitor tablet
    28 μl of 0.5 M PMSF in DMSO
    4.2 ml of H2O
  5. 20 ml of IP buffer with 0.1% digitonin
    10 ml of 2x IP buffer
    0.2 ml of 10% digitonin
    9.8 ml of H2O
    Protease inhibitors (20 μl of PMSF, leupeptin, and pepstatin)
  6. 40 ml of IP buffer with 0.1% digitonin
    20 ml of 2x IP buffer
    0.4 ml of 10% digitonin
    19.6 ml of H2O
    Protease inhibitors (40 μl of PMSF, leupeptin, and pepstatin)
  7. 150 μl of elution buffer with 1μg/μl 3x FLAG peptide
    75 μl of 2x IP buffer
    3.75 μl of 10% digitonin
    30 μl of 5 mg/ml 3x FLAG peptide
    41.25 μl of H2O
  8. To make 150 μl of elution buffer with 0.1 μg/μl 3x FLAG peptide
    75 μl of 2x IP buffer
    3.75 μl of 10% digitonin
    3 μl of 5 mg/ml 3x FLAG peptide
    68.25 μl of H2O

Acknowledgments

This protocol has been modified and adapted in the Espenshade Lab, Johns Hopkins School of Medicine. Funding to support different projects that have used this protocol has come from NIH – National Heart, Lung, and Blood Institute, National Institute of Allergy and Infectious Diseases, the Pancreatic Cancer Action Network, and the American Heart Association.

References

  1. Breslow, D. K., Collins, S. R., Bodenmiller, B., Aebersold, R., Simons, K., Shevchenko, A., Ejsing, C. S. and Weissman, J. S. (2010). Orm family proteins mediate sphingolipid homeostasis. Nature 463(7284): 1048-1053.


How to cite this protocol: Tong, Z. (2011). Large Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast. Bio-protocol Bio101: e12. DOI: 10.21769/BioProtoc.12; Full Text



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