Knight:Protein solubility

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==References==
==References==
*This protocol is derived from a talk with [[User:Kathmc|Kathleen]] and modified according to how I've been doing my protein purifications and running gels to date.
*This protocol is derived from a talk with [[User:Kathmc|Kathleen]] and modified according to how I've been doing my protein purifications and running gels to date.
 +
<biblio>
 +
#Marblestone-ProtSci-2006 pmid=16322573
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</biblio>

Revision as of 13:44, 14 December 2006

in progress. may contain errors!

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
    1. Pellet cells by spinning at 4000 x g for 15 mins at 4°C.
    2. Resuspend in 50 μL denaturing lysis buffer.
    3. 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?
    4. Centrifuge lysate at 10000 x g for 30 mins at room temperature.
      • 10 mins might be enough.
    5. Save 13 μL to run on a gel. (This is the total protein.)
  3. Take another 2mL aliquot of culture and
    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. Optional: Add 1% Triton X-100 (v/v) and incubate for 1 hr at 4 °C [1]
      • Might help keep the cellular proteins in the soluble fraction. Most of the cellular protein appear to come out in the insoluble fraction which they shouldn't.
    6. Centrifuge lysate at 10000 x g for 30 mins at 4°C.
      • 10 mins might be enough.
    7. Save 13 μL of supernatant to run on a gel. (This is the soluble fraction).
    8. Resuspend pellet in 50 μL denaturing lysis buffer.
    9. Centrifuge at 10000 x g for 20 mins at 4°C.
    10. 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 the "denaturing" lysis protocol above. Thus, the freeze thaw method may result in more complete cell lysis.

References

  • 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.
  1. Marblestone JG, Edavettal SC, Lim Y, Lim P, Zuo X, and Butt TR. . pmid:16322573. PubMed HubMed [Marblestone-ProtSci-2006]
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