Knight:Protein solubility

Overview
This is a quick protocol to assess how soluble a particular protein is.

Materials

 * Denaturing lysis buffer
 * Native lysis buffer
 * Triton X-100
 * 2 mL centrifuge tubes
 * Centrifuge

Procedure

 * 1) Grow a 6mL culture.
 * 2) Take 2mL of culture and move to 2mL centrifuge tube.
 * 3) Pellet cells by spinning at 4000 x g for 15 mins at 4&deg;C.
 * 4) Resuspend in 50 &mu;L denaturing lysis buffer + 2% SDS.
 * 5) Freeze the cells at -80&deg;C and thaw for 3 cycles.
 * 6) *To speed things up, try quick freezing in an ethanol-dry ice bath and thaw on slushy ice.
 * 7) Add 1% Triton X-100 (v/v) Marblestone-ProtSci-2006
 * 8) *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.
 * 9) Incubate cells with agitation for 1 hr at room temperature.
 * 10) *Use an orbis shaker on the bench to do this temp (usually kept in 37&deg; incubator). Note that the shaker moves during shaking.
 * 11) *Kathleen suggests just lysing by heating at 90&deg;C for 10 mins but this may require the presence of SDS loading buffer?
 * 12) Centrifuge lysate at 10000 x g for 30 mins at room temperature.
 * 13) *10 mins might be enough.
 * 14) Save 13 &mu;L to run on a gel. (This is the total protein.)
 * 15) Take another 2mL aliquot of culture and move to 2 mL centrifuge tube
 * 16) Pellet cells by spinning at 4000 x g for 15 mins at 4&deg;C.
 * 17) Resuspend in 50 &mu;L of native lysis buffer.
 * 18) Optional: Add 0.5 &mu;L 100 mg/mL lysozyme to 1 mg/mL final concentration.
 * 19) *Note that lysozyme is ~14 kDa so it will run close to my protein on a gel! Kathleen says if it is going to be a problem, freeze-thaw only should work reasonably well for this test. It is hard to sonicate small volumes. Could also try a commercial "mild lysis" reagent, although people in the Sauer lab have had varied success with these.
 * 20) Freeze the cells at -80&deg;C and thaw for 3 cycles.
 * 21) *To speed things up, try quick freezing in an ethanol-dry ice bath and thaw on slushy ice.
 * 22) Add 1% Triton X-100 (v/v) Marblestone-ProtSci-2006
 * 23) *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.
 * 24) Incubate for 1 hr at 4 &deg;C
 * 25) Centrifuge lysate at 10000 x g for 30 mins at 4&deg;C.
 * 26) *10 mins might be enough.
 * 27) Save 13 &mu;L of supernatant to run on a gel. (This is the soluble fraction).
 * 28) Resuspend pellet in 50 &mu;L denaturing lysis buffer + 2% SDS.
 * 29) *Letting this incubate at room temperature with agitation for 20 minutes decreases the viscosity of this fraction (facilitating gel loading).
 * 30) Centrifuge at 10000 x g for 20 mins at 4&deg;C.
 * 31) Save 13 &mu;L resuspended pellet to load on a gel. (This is the insoluble fraction).