Drummond:Solubility

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==Introduction==
==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.
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.
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#Grow a 6mL overnight culture.
#Grow a 6mL overnight culture.
#Take 2mL of culture and move to 2mL centrifuge tube.
#Take 2mL of culture and move to 2mL centrifuge tube.
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#Pellet cells by spinning at 4000 x ''g'' for 15 mins at 4&deg;C.
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#Pellet cells by spinning at 20000 x ''g'' for 15 seconds.  Discard supernatant.
#Resuspend in 100 &mu;L [[Drummond:Solubility#Materials|solubilization buffer]].
#Resuspend in 100 &mu;L [[Drummond:Solubility#Materials|solubilization buffer]].
#Lyse cells
#Lyse cells
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#*''Use glass-bead protocol, e.g. [[Silver:_Lysate_for_Western|Silver Lab yeast lysis]]''
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#*''Use chemical lysis, e.g. [http://www.emdbiosciences.com/html/NVG/yeastbuster.htm YeastBuster]''
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#Add 1% Triton X-100 (v/v) <cite>Marblestone-ProtSci-2006</cite>
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#*''Helps to keep the cellular proteins in the soluble fraction. Otherwise, most of the cellular protein appears to come out in the insoluble fraction without this step which it shouldn't.''
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#Incubate cells with agitation for 1 hr at room temperature.
#Incubate cells with agitation for 1 hr at room temperature.
#Centrifuge lysate at 10000 x ''g'' for 30 mins at room temperature.
#Centrifuge lysate at 10000 x ''g'' for 30 mins at room temperature.
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Soluble and insoluble fractions:
Soluble and insoluble fractions:
#Take a 2mL aliquot of culture and move to 2 mL centrifuge tube
#Take a 2mL aliquot of culture and move to 2 mL centrifuge tube
-
#Pellet cells by spinning at 4000 x ''g'' for 15 mins at 4&deg;C.
+
#Pellet cells by spinning at 20000 x ''g'' for 15 seconds. Discard supernatant.
#Resuspend in 100 &mu;L [[Drummond:Solubility#Materials|suspension buffer]]
#Resuspend in 100 &mu;L [[Drummond:Solubility#Materials|suspension buffer]]
#Lyse cells
#Lyse cells
-
#*''Use glass-bead protocol, e.g. [[Silver:_Lysate_for_Western|Silver Lab yeast lysis]]''
+
#*''Use chemical lysis, e.g. [http://www.emdbiosciences.com/html/NVG/yeastbuster.htm YeastBuster]''
-
#Add 1% Triton X-100 (v/v) <cite>Marblestone-ProtSci-2006</cite>
+
#Incubate cells with agitation for 1 hr at room temperature.
-
#*''Helps to keep the cellular proteins in the soluble fraction. Otherwise, most of the cellular protein appears to come out in the insoluble fraction without this step which it shouldn't.''
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#Incubate for 1 hr at 4 &deg;C
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#Centrifuge lysate at 10000 x ''g'' for 30 mins at 4&deg;C.
#Centrifuge lysate at 10000 x ''g'' for 30 mins at 4&deg;C.
#*''10 mins might be enough.''
#*''10 mins might be enough.''
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* 20 mM [[phosphate buffer]], pH 8.0
* 20 mM [[phosphate buffer]], pH 8.0
* 300 mM NaCL
* 300 mM NaCL
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* 8 M urea (a strong denaturant)
 
* 2% v/v sodium dodecyl sulfate (SDS, an ionic surfactant, or detergent)
* 2% v/v sodium dodecyl sulfate (SDS, an ionic surfactant, or detergent)
* 2mM dithiothreitol (DTT, a reducing agent)
* 2mM dithiothreitol (DTT, a reducing agent)
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Alternatives:
Alternatives:
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* 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 <cite>Marblestone-ProtSci-2006</cite>
*50 mM CAPS at pH 11, 0.3 M NaCl, 0.3% N-lauryl sarcosine, and 1 mM DTT <cite>Marblestone-ProtSci-2006</cite>
**[http://www.sigmaaldrich.com/catalog/search/ProductDetail/ALDRICH/163767 CAPS, Aldrich]
**[http://www.sigmaaldrich.com/catalog/search/ProductDetail/ALDRICH/163767 CAPS, Aldrich]
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#Collins-EMBOJ-2005 pmid=15889152  
#Collins-EMBOJ-2005 pmid=15889152  
</biblio>
</biblio>
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Revision as of 19:03, 6 July 2007

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Contents

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
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