Drummond:Solubility

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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 &mu;L solubilization buffer.
 * 5) Lyse cells
 * 6) *Use chemical lysis, e.g. YeastBuster
 * 7) Incubate cells with agitation for 1 hr at room temperature.
 * 8) Centrifuge lysate at 10000 x g for 30 mins at room temperature.
 * 9) *10 mins might be enough.
 * 10) 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 &mu;L suspension buffer
 * 5) Lyse cells
 * 6) *Add 200 &mu;L YeastBuster lysis reagent + 1X protease inhibitors
 * 7) *Incubate cells with gentle agitation for 20 min at room temperature.
 * 8) Centrifuge lysate at 10000 x g for 10 mins at 4&deg;C.
 * 9) Draw off and save supernatant. (This is the soluble fraction).
 * 10) Wash pellet 2X with 500 &mu;L water.
 * 11) Resuspend pellet in 100 &mu;L solubilization buffer.
 * 12) Centrifuge at 10000 x g for 20 mins at 4&deg;C.
 * 13) Draw off and save supernatant. (This is the insoluble fraction).

Suspension buffer
Keys: pH buffering, light detergent, protease inhibitors (roughly from Ripaud-EMBOJ-2003 )
 * 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)

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
 * H2O to 1 mL (810 &mu;L)

Alternatives:
 * 3 mL of PBS (pH 8.0), 300 mM NaCl, 10 mM imidazole Marblestone-ProtSci-2006

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 &mu;L phosphate buffer, pH 8.0
 * 60 &mu;L 5 M NaCl
 * 100 &mu;L 20% SDS
 * 100 &mu;L 20 mM DTT
 * 100 &mu;L 10% Triton X-100
 * 1X protease inhibitor cocktail -- 50 &mu;L of Sigma's fungal protease inhibitor cocktail
 * H2O to 1 mL (390 &mu;L)

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 Marblestone-ProtSci-2006
 * CAPS, Aldrich
 * 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 Mechin-Prot-2003
 * 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) Collins-EMBOJ-2005

Links to protocols
Knight:Protein solubility