Drummond:Solubility: Difference between revisions
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==Introduction== | ==Introduction== | ||
Goal: to measure the proportion of a protein in the soluble versus insoluble state. | 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== | ==Principle== | ||
The basic method | 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: | 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. | **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. | **Control: Do the lysis in solubilization buffer, and save that fraction as total protein. Compare total protein to soluble + insoluble protein. | ||
Line 17: | Line 19: | ||
Total protein: | Total protein: | ||
#Grow a 6mL overnight culture. | #Grow a 6mL overnight culture. | ||
# | #Move 2mL of culture into a 2mL microcentrifuge tube. | ||
#Pellet cells by spinning at | #Pellet cells by spinning at 20000 x ''g'' for 15 seconds. Discard supernatant. | ||
#Resuspend in 100 μL [[Drummond:Solubility#Materials|solubilization buffer]]. | #Resuspend in 100 μL [[Drummond:Solubility#Materials|solubilization buffer]]. | ||
#Lyse cells | #Lyse cells | ||
#*''Use | #*''Use chemical lysis, e.g. [http://www.emdbiosciences.com/html/NVG/yeastbuster.htm YeastBuster]'' | ||
#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. | ||
Line 30: | Line 30: | ||
Soluble and insoluble fractions: | Soluble and insoluble fractions: | ||
# | #Grow a 6mL overnight culture. | ||
#Pellet cells by spinning at | #Move 2mL of culture into a 2mL microcentrifuge tube. | ||
#Pellet cells by spinning at 20000 x ''g'' for 15 seconds. Discard supernatant. | |||
#Resuspend in 100 μL [[Drummond:Solubility#Materials|suspension buffer]] | #Resuspend in 100 μL [[Drummond:Solubility#Materials|suspension buffer]] | ||
#Lyse cells | #Lyse cells | ||
#* | #*Add 200 μL [http://www.emdbiosciences.com/html/NVG/yeastbuster.htm YeastBuster] lysis reagent + 1X protease inhibitors | ||
#*Incubate cells with gentle agitation for 20 min at room temperature. | |||
#Centrifuge lysate at 10000 x ''g'' for 10 mins at 4°C. | |||
#Incubate for | |||
#Centrifuge lysate at 10000 x ''g'' for | |||
#Draw off and save supernatant. (This is the soluble fraction). | #Draw off and save supernatant. (This is the soluble fraction). | ||
#Wash pellet 2X with 500 μL water. | |||
#Resuspend pellet in 100 μL [[Drummond:Solubility#Materials|solubilization buffer]]. | #Resuspend pellet in 100 μL [[Drummond:Solubility#Materials|solubilization buffer]]. | ||
#Centrifuge at 10000 x ''g'' for 20 mins at 4°C. | #Centrifuge at 10000 x ''g'' for 20 mins at 4°C. | ||
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*PBS, pH 8.0 | *PBS, pH 8.0 | ||
*100 mM NaCl, | *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) | *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 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 | |||
*H<sub>2</sub>O to 1 mL (810 μL) | |||
Alternatives: | Alternatives: | ||
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* 20 mM [[phosphate buffer]], pH 8.0 | * 20 mM [[phosphate buffer]], pH 8.0 | ||
* 300 mM | * 300 mM NaCl | ||
* 2% v/v sodium dodecyl sulfate (SDS, an ionic surfactant, or detergent) | * 2% v/v sodium dodecyl sulfate (SDS, an ionic surfactant, or detergent) | ||
* | * 2 mM 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) | * 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 | * 1% v/v Triton X-100 | ||
To make 1 mL (enough to process 9-10 pellets from 2mL saturated cultures): | |||
* 200 μL [[phosphate buffer]], pH 8.0 | |||
* 60 μL 5 M NaCl | |||
* 100 μL 20% SDS | |||
* 100 μL 20 mM DTT | |||
* 100 μL 10% Triton X-100 | |||
* 1X protease inhibitor cocktail -- 50 μL of Sigma's fungal protease inhibitor cocktail | |||
* H<sub>2</sub>O to 1 mL (390 μL) | |||
Alternatives: | 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 <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?ProdNo=L7414&Brand=SIGMA N-lauroylsarcosine, Sigma] | |||
*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<cite>Mechin-Prot-2003</cite> | *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<cite>Mechin-Prot-2003</cite> | ||
*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) <cite>Collins-EMBOJ-2005</cite> | *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) <cite>Collins-EMBOJ-2005</cite> | ||
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#Collins-EMBOJ-2005 pmid=15889152 | #Collins-EMBOJ-2005 pmid=15889152 | ||
</biblio> | </biblio> | ||
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Latest revision as of 09:47, 8 July 2007
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:
- 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:
- Grow a 6mL overnight culture.
- Move 2mL of culture into a 2mL microcentrifuge tube.
- Pellet cells by spinning at 20000 x g for 15 seconds. Discard supernatant.
- Resuspend in 100 μL solubilization buffer.
- Lyse cells
- Use chemical lysis, e.g. YeastBuster
- Incubate cells with agitation for 1 hr at room temperature.
- Centrifuge lysate at 10000 x g for 30 mins at room temperature.
- 10 mins might be enough.
- Draw off and save supernatant. (This is the total protein fraction.)
Soluble and insoluble fractions:
- Grow a 6mL overnight culture.
- Move 2mL of culture into a 2mL microcentrifuge tube.
- Pellet cells by spinning at 20000 x g for 15 seconds. Discard supernatant.
- Resuspend in 100 μL suspension buffer
- Lyse cells
- Add 200 μL YeastBuster lysis reagent + 1X protease inhibitors
- Incubate cells with gentle agitation for 20 min at room temperature.
- Centrifuge lysate at 10000 x g for 10 mins at 4°C.
- Draw off and save supernatant. (This is the soluble fraction).
- Wash pellet 2X with 500 μL water.
- Resuspend pellet in 100 μL solubilization buffer.
- Centrifuge at 10000 x g for 20 mins at 4°C.
- 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
- H2O to 1 mL (810 μL)
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)
- 2 mM 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
To make 1 mL (enough to process 9-10 pellets from 2mL saturated cultures):
- 200 μL phosphate buffer, pH 8.0
- 60 μL 5 M NaCl
- 100 μL 20% SDS
- 100 μL 20 mM DTT
- 100 μL 10% Triton X-100
- 1X protease inhibitor cocktail -- 50 μL of Sigma's fungal protease inhibitor cocktail
- H2O to 1 mL (390 μL)
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
- Urea should always be freshly prepared and deionized just prior to use.
Links to protocols
References
- Ripaud L, Maillet L, and Cullin C. The mechanisms of [URE3] prion elimination demonstrate that large aggregates of Ure2p are dead-end products. EMBO J. 2003 Oct 1;22(19):5251-9. DOI:10.1093/emboj/cdg488 |
- Marblestone JG, Edavettal SC, Lim Y, Lim P, Zuo X, and Butt TR. Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein Sci. 2006 Jan;15(1):182-9. DOI:10.1110/ps.051812706 |
- Méchin V, Consoli L, Le Guilloux M, and Damerval C. An efficient solubilization buffer for plant proteins focused in immobilized pH gradients. Proteomics. 2003 Jul;3(7):1299-302. DOI:10.1002/pmic.200300450 |
- Collins KM, Thorngren NL, Fratti RA, and Wickner WT. Sec17p and HOPS, in distinct SNARE complexes, mediate SNARE complex disruption or assembly for fusion. EMBO J. 2005 May 18;24(10):1775-86. DOI:10.1038/sj.emboj.7600658 |