Knight:Protein solubility

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(Procedure)
 
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<font color=red>in progress.  may contain errors!</font>
 
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==Overview==
==Overview==
This is a quick protocol to assess how soluble a particular protein is.
This is a quick protocol to assess how soluble a particular protein is.
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*[[Knight:Purification of His-tagged proteins/Denaturing#Lysis and column equilibration buffer (Qiagen buffer B)|Denaturing lysis buffer]]
*[[Knight:Purification of His-tagged proteins/Denaturing#Lysis and column equilibration buffer (Qiagen buffer B)|Denaturing lysis buffer]]
*[[Knight:Purification of His-tagged proteins/Native#Lysis and column equilibration buffer|Native lysis buffer]]
*[[Knight:Purification of His-tagged proteins/Native#Lysis and column equilibration buffer|Native lysis buffer]]
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*Triton X-100
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*2 mL centrifuge tubes
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*Centrifuge
==Procedure==
==Procedure==
#Grow a 6mL culture.
#Grow a 6mL culture.
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#Take 2mL of culture and
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#Take 2mL of culture and move to 2mL centrifuge tube.
##Pellet cells by spinning at 4000 x ''g'' for 15 mins at 4&deg;C.
##Pellet cells by spinning at 4000 x ''g'' for 15 mins at 4&deg;C.
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##Resuspend in 50 &mu;L [[Knight:Purification of His-tagged proteins/Denaturing#Lysis and column equilibration buffer (Qiagen buffer B)|denaturing lysis buffer]].
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##Resuspend in 50 &mu;L [[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&deg;C and thaw for 3 cycles.
##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.''
##*''To speed things up, try quick freezing in an ethanol-dry ice bath and thaw on slushy ice.''
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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.''
##Incubate cells with agitation for 1 hr at room temperature.
##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.''
##*''Use an orbis shaker on the bench to do this temp (usually kept in 37&deg; incubator).  Note that the shaker moves during shaking.''
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##*''10 mins might be enough.''
##*''10 mins might be enough.''
##Save 13 &mu;L to run on a gel.  (This is the total protein.)
##Save 13 &mu;L to run on a gel.  (This is the total protein.)
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#Take another 2mL aliquot of culture and
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#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.
##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]].
##Resuspend in 50 &mu;L of [[Knight:Purification of His-tagged proteins/Native#Lysis and column equilibration buffer|native lysis buffer]].
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##Freeze the cells at -80&deg;C and thaw for 3 cycles.
##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.''
##*''To speed things up, try quick freezing in an ethanol-dry ice bath and thaw on slushy ice.''
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##Optional: Add 1% Triton X-100 (v/v) and incubate for 1 hr at 4 &deg;C <cite>Marblestone-ProtSci-2006</cite>
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##Add 1% Triton X-100 (v/v) <cite>Marblestone-ProtSci-2006</cite>
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##*''Might help keep the cellular proteins in the soluble fraction.  Most of the cellular protein appear to come out in the insoluble fraction without this step which it shouldn't.''
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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 for 1 hr at 4 &deg;C
##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.''
##Save 13 &mu;L of supernatant to run on a gel. (This is the soluble fraction).
##Save 13 &mu;L of supernatant to run on a gel. (This is the soluble fraction).
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##Resuspend pellet in 50 &mu;L [[Knight:Purification of His-tagged proteins/Denaturing#Lysis and column equilibration buffer (Qiagen buffer B)|denaturing lysis buffer]].
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##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.
 +
##*''Letting this incubate at room temperature with agitation for 20 minutes decreases the viscosity of this fraction (facilitating gel loading).''
##Centrifuge at 10000 x ''g'' for 20 mins at 4&deg;C.
##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).
##Save 13 &mu;L resuspended pellet to load on a gel.  (This is the insoluble fraction).
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#Marblestone-ProtSci-2006 pmid=16322573
#Marblestone-ProtSci-2006 pmid=16322573
</biblio>
</biblio>
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[[Category:Protocol]] [[Category:Protein]] [[Category:In vitro]]

Current revision

Contents

Overview

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

Materials

Procedure

  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.
      • Letting this incubate at room temperature with agitation for 20 minutes decreases the viscosity of this fraction (facilitating gel loading).
    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).

Notes

  • The amount of material you load from the supernatant and pellet should add up to the total protein so that you are comparing equivalent amounts.
  • Tom pointed out that a confounding factor is how well my cells lyse in the "native" versus "denatured" conditions. If my cells don't lyse in the native conditions then my protein will end up in the pellet even if they are soluble. However, it turns out that the "native" lysis protocol above seems to be more efficient than typical "denaturing" lysis (add denaturing lysis buffer and incubate with shaking for 1 hr at room temperature). Thus, the freeze thaw method may result in more complete cell lysis.

References

  1. Marblestone JG, Edavettal SC, Lim Y, Lim P, Zuo X, and Butt TR. . pmid:16322573. PubMed HubMed [Marblestone-ProtSci-2006]
  2. This protocol is derived from a talk with Kathleen and modified according to how I've been doing my protein purifications and running gels to date. [Kathleen]
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