Drummond:Solubility

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(update - chemical lysis)
(to make 1ml)
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*PBS, pH 8.0
*PBS, pH 8.0
*100 mM NaCl,  
*100 mM NaCl,  
-
*1x protease inhibitor cocktail (0.46 mug/ml leupeptin, 3.5 mug/ml pepstatin, 2.4 mug/ml pefabloc-SC, 1 mM PMSF)
 
*0.2% v/v Triton X-100
*0.2% v/v Triton X-100
 +
*1x protease inhibitor cocktail (0.46 mug/ml leupeptin, 3.5 mug/ml pepstatin, 2.4 mug/ml pefabloc-SC, 1 mM PMSF)
(roughly from <cite>Ripaud-EMBOJ-2003</cite>)
(roughly from <cite>Ripaud-EMBOJ-2003</cite>)
 +
 +
To make 1 mL (enough to process 9-10 pellets from 2mL saturated cultures):
 +
*100 &mu;L 10X PBS
 +
*20 &mu;L 5 M NaCl
 +
*20 &mu;L 10% v/v Triton X-100
 +
*1X protease inhibitor cocktail -- 50 &mu;L of Sigma's fungal protease inhibitor cocktail
Alternatives:
Alternatives:

Revision as of 12:39, 8 July 2007

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Introduction

Goal: to measure the proportion of a protein in the soluble versus insoluble state. The standard method uses antibody probes against protein extracts from the supernatant and pellet of an aqueous lysis.

Principle

The basic method is to lyse cells into an aqueous buffer, spin down the cell debris, pull off the supernatant and store it as the soluble fraction, then solubilize proteins remaining in the pellet using a solubilization buffer containing various detergents and denaturing agents (e.g. SDS, urea), spin down the cell debris again, and pull off the supernatant and store it as the insoluble fraction.

Questions:

  1. How do you ensure that you've preserved the composition of total protein in each fraction?
    • Extract in the same amount of buffer in each case, and load identical amounts of each fraction.
    • Control: Do the lysis in solubilization buffer, and save that fraction as total protein. Compare total protein to soluble + insoluble protein.

Protocol

(Adapted from Knight:Protein solubility, a bacterial protocol. Here, the organisms is assumed to be S. cerevisiae.)

Total protein:

  1. Grow a 6mL overnight culture.
  2. Move 2mL of culture into a 2mL microcentrifuge tube.
  3. Pellet cells by spinning at 20000 x g for 15 seconds. Discard supernatant.
  4. Resuspend in 100 μL solubilization buffer.
  5. Lyse cells
  6. Incubate cells with agitation for 1 hr at room temperature.
  7. Centrifuge lysate at 10000 x g for 30 mins at room temperature.
    • 10 mins might be enough.
  8. Draw off and save supernatant. (This is the total protein fraction.)

Soluble and insoluble fractions:

  1. Grow a 6mL overnight culture.
  2. Move 2mL of culture into a 2mL microcentrifuge tube.
  3. Pellet cells by spinning at 20000 x g for 15 seconds. Discard supernatant.
  4. Resuspend in 100 μL suspension buffer
  5. Lyse cells
    • Add 200 μL YeastBuster lysis reagent + 1X protease inhibitors
    • Incubate cells with gentle agitation for 20 min at room temperature.
  6. Centrifuge lysate at 10000 x g for 10 mins at 4°C.
  7. Draw off and save supernatant. (This is the soluble fraction).
  8. Wash pellet 2X with 500 μL water.
  9. Resuspend pellet in 100 μL solubilization buffer.
  10. Centrifuge at 10000 x g for 20 mins at 4°C.
  11. Draw off and save supernatant. (This is the insoluble fraction).

Materials

Suspension buffer

Keys: pH buffering, light detergent, protease inhibitors

  • PBS, pH 8.0
  • 100 mM NaCl,
  • 0.2% v/v Triton X-100
  • 1x protease inhibitor cocktail (0.46 mug/ml leupeptin, 3.5 mug/ml pepstatin, 2.4 mug/ml pefabloc-SC, 1 mM PMSF)

(roughly from [1])

To make 1 mL (enough to process 9-10 pellets from 2mL saturated cultures):

  • 100 μL 10X PBS
  • 20 μL 5 M NaCl
  • 20 μL 10% v/v Triton X-100
  • 1X protease inhibitor cocktail -- 50 μL of Sigma's fungal protease inhibitor cocktail

Alternatives:

  • 3 mL of PBS (pH 8.0), 300 mM NaCl, 10 mM imidazole [2]

Solubilization buffer

Keys: pH buffering, reducing agent, strong chaotropic (denaturing) agent, strong detergent

  • 20 mM phosphate buffer, pH 8.0
  • 300 mM NaCL
  • 2% v/v sodium dodecyl sulfate (SDS, an ionic surfactant, or detergent)
  • 2mM dithiothreitol (DTT, a reducing agent)
  • 1x protease inhibitor cocktail (0.46 mug/ml leupeptin, 3.5 mug/ml pepstatin, 2.4 mug/ml pefabloc-SC, 1 mM PMSF)
  • 1% v/v Triton X-100

Alternatives:

  • 20 mM phosphate buffer, pH 8.0, 300 mM NaCL, 8 M urea (a strong denaturant), 2% v/v sodium dodecyl sulfate (SDS, an ionic surfactant, or detergent), 2mM dithiothreitol (DTT, a reducing agent), 1x protease inhibitor cocktail (0.46 mug/ml leupeptin, 3.5 mug/ml pepstatin, 2.4 mug/ml pefabloc-SC, 1 mM PMSF), 1% v/v Triton X-100
  • 50 mM CAPS at pH 11, 0.3 M NaCl, 0.3% N-lauryl sarcosine, and 1 mM DTT [2]
  • 5 M urea, 2 M thiourea, 2% 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, 2% N-decyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate, 20 mM dithiothreitol, 5 mM Tris(2-carboxyethyl) phosphine[3]
  • 20 mM HEPES/KOH, pH 7.4, 100 mM NaCl, 2 mM EDTA, 0.5% Triton X-100 (Anatrace), 20% glycerol, 1 times protease inhibitor cocktail (0.46 mug/ml leupeptin, 3.5 mug/ml pepstatin, 2.4 mug/ml pefabloc-SC, 1 mM PMSF) [4]

Notes

  1. Urea should always be freshly prepared and deionized just prior to use.

Links to protocols

Knight:Protein solubility

References

  1. Ripaud L, Maillet L, and Cullin C. . pmid:14517262. PubMed HubMed [Ripaud-EMBOJ-2003]
  2. Marblestone JG, Edavettal SC, Lim Y, Lim P, Zuo X, and Butt TR. . pmid:16322573. PubMed HubMed [Marblestone-ProtSci-2006]
  3. Méchin V, Consoli L, Le Guilloux M, and Damerval C. . pmid:12872230. PubMed HubMed [Mechin-Prot-2003]
  4. Collins KM, Thorngren NL, Fratti RA, and Wickner WT. . pmid:15889152. PubMed HubMed [Collins-EMBOJ-2005]
All Medline abstracts: PubMed HubMed
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