Drummond:Solubility

From OpenWetWare

Revision as of 19:12, 6 July 2007 by Dadrummond (Talk | contribs)
Jump to: navigation, search
the drummond lab

home      people      research      publications      news      jobs      protocols      contact     


Introduction

Goal: to measure the proportion of a protein in the soluble versus insoluble state. Typical assays seem to use antibody probes against the supernatant and pellet of a standard lysis.

Principle

The basic method of all assays I've seen is to lyse cells into an aqueous buffer, spin down the pellet, 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 pellet again, and pull off the supernatant and store it as the insoluble fraction.

Questions:

  • 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. Take 2mL of culture and move to 2mL centrifuge 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. Take a 2mL aliquot of culture and move to 2 mL centrifuge tube
  2. Pellet cells by spinning at 20000 x g for 15 seconds. Discard supernatant.
  3. Resuspend in 100 μL suspension buffer
  4. Lyse cells
  5. Incubate cells with agitation for 1 hr at room temperature.
  6. Centrifuge lysate at 10000 x g for 30 mins at 4°C.
    • 10 mins might be enough.
  7. Draw off and save supernatant. (This is the soluble fraction).
  8. Resuspend pellet in 100 μL solubilization buffer.
  9. Centrifuge at 10000 x g for 20 mins at 4°C.
  10. 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,
  • 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

(roughly from [1])

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
Personal tools