Drummond:Solubility

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(principle, and some questions answered)
(Principle: protocol)
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==Principle==
==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 (e.g. Triton X-100), spin down the pellet again, and pull off the supernatant and store it as the insoluble fraction.
+
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?  Answer: 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.
Questions: How do you ensure that you've preserved the composition of total protein in each fraction?  Answer: 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.
 +
 +
==Protocol==
 +
(Adapted from [[Knight:Protein solubility]].)
 +
#Grow a 6mL culture.
 +
#Take 2mL of culture and move to 2mL centrifuge tube.
 +
##Pellet cells by spinning at 4000 x ''g'' for 15 mins at 4°C.
 +
##Resuspend in 50 μL [[Knight:Purification of His-tagged proteins/Denaturing#Lysis and column equilibration buffer (Qiagen buffer B)|denaturing lysis buffer]] + 2% SDS.
 +
##Freeze the cells at -80°C and thaw for 3 cycles.
 +
##*''To speed things up, try quick freezing in an ethanol-dry ice bath and thaw on slushy ice.''
 +
##Add 1% Triton X-100 (v/v) <cite>Marblestone-ProtSci-2006</cite>
 +
##*''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.''
 +
##Incubate cells with agitation for 1 hr at room temperature.
 +
##*''Use an orbis shaker on the bench to do this temp (usually kept in 37&deg; incubator).  Note that the shaker moves during shaking.''
 +
##*''Kathleen suggests just lysing by heating at 90&deg;C for 10 mins but this may require the presence of SDS loading buffer?''
 +
##Centrifuge lysate at 10000 x ''g'' for 30 mins at room temperature.
 +
##*''10 mins might be enough.''
 +
##Save 13 &mu;L to run on a gel.  (This is the total protein.)
 +
#Take another 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.
 +
##Resuspend in 50 &mu;L of [[Knight:Purification of His-tagged proteins/Native#Lysis and column equilibration buffer|native lysis buffer]].
 +
##Optional: Add 0.5 &mu;L 100 mg/mL lysozyme to 1 mg/mL final concentration.
 +
##*''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.''
 +
##Freeze the cells at -80&deg;C and thaw for 3 cycles.
 +
##*''To speed things up, try quick freezing in an ethanol-dry ice bath and thaw on slushy ice.''
 +
##Add 1% Triton X-100 (v/v) <cite>Marblestone-ProtSci-2006</cite>
 +
##*''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.''
 +
##Incubate for 1 hr at 4 &deg;C
 +
##Centrifuge lysate at 10000 x ''g'' for 30 mins at 4&deg;C.
 +
##*''10 mins might be enough.''
 +
##Save 13 &mu;L of supernatant to run on a gel. (This is the soluble fraction).
 +
##Resuspend pellet in 50 &mu;L [[Knight:Purification of His-tagged proteins/Denaturing#Lysis and column equilibration buffer (Qiagen buffer B)|denaturing lysis buffer]] + 2% SDS.
 +
##Centrifuge at 10000 x ''g'' for 20 mins at 4&deg;C.
 +
##Save 13 &mu;L resuspended pellet to load on a gel.  (This is the insoluble fraction).
==Links to protocols==
==Links to protocols==

Revision as of 12:03, 19 June 2007

Contents

Introduction

I'd like 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? Answer: 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.

Protocol

(Adapted from Knight:Protein solubility.)

  1. Grow a 6mL culture.
  2. Take 2mL of culture and move to 2mL centrifuge tube.
    1. Pellet cells by spinning at 4000 x g for 15 mins at 4°C.
    2. Resuspend in 50 μL denaturing lysis buffer + 2% SDS.
    3. Freeze the cells at -80°C and thaw for 3 cycles.
      • To speed things up, try quick freezing in an ethanol-dry ice bath and thaw on slushy ice.
    4. Add 1% Triton X-100 (v/v) [1]
      • 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.
    5. Incubate cells with agitation for 1 hr at room temperature.
      • Use an orbis shaker on the bench to do this temp (usually kept in 37° incubator). Note that the shaker moves during shaking.
      • Kathleen suggests just lysing by heating at 90°C for 10 mins but this may require the presence of SDS loading buffer?
    6. Centrifuge lysate at 10000 x g for 30 mins at room temperature.
      • 10 mins might be enough.
    7. Save 13 μL to run on a gel. (This is the total protein.)
  3. Take another 2mL aliquot of culture and move to 2 mL centrifuge tube
    1. Pellet cells by spinning at 4000 x g for 15 mins at 4°C.
    2. Resuspend in 50 μL of native lysis buffer.
    3. Optional: Add 0.5 μL 100 mg/mL lysozyme to 1 mg/mL final concentration.
      • 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.
    4. Freeze the cells at -80°C and thaw for 3 cycles.
      • To speed things up, try quick freezing in an ethanol-dry ice bath and thaw on slushy ice.
    5. Add 1% Triton X-100 (v/v) [1]
      • 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.
    6. Incubate for 1 hr at 4 °C
    7. Centrifuge lysate at 10000 x g for 30 mins at 4°C.
      • 10 mins might be enough.
    8. Save 13 μL of supernatant to run on a gel. (This is the soluble fraction).
    9. Resuspend pellet in 50 μL denaturing lysis buffer + 2% SDS.
    10. Centrifuge at 10000 x g for 20 mins at 4°C.
    11. Save 13 μL resuspended pellet to load on a gel. (This is the insoluble fraction).

Links to protocols

Knight:Protein solubility

References

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