Drummond:Solubility

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(protocol improvements)
(Protocol)
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==Protocol==
==Protocol==
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(Adapted from [[Knight:Protein solubility]], a bacterial protocol.  Here, the organisms asssumed to be <i>S. cerevisiae</i>.)
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(Adapted from [[Knight:Protein solubility]], a bacterial protocol.  Here, the organisms is assumed to be <i>S. cerevisiae</i>.)
 +
 
Total protein:
Total protein:
#Grow a 6mL overnight culture.
#Grow a 6mL overnight culture.
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#Take a 2mL aliquot of culture and move to 2 mL centrifuge tube
#Take a 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.
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#Resuspend in 100 &mu;L of [[#Materials/.
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#Resuspend in 100 &mu;L [[Drummond:Solubility#Materials|native buffer]]
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#Lyse  
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#Lyse cells
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#*''Use glass-bead protocol''
#Add 1% Triton X-100 (v/v) <cite>Marblestone-ProtSci-2006</cite>
#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.''
#*''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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#*''10 mins might be enough.''
#*''10 mins might be enough.''
#Draw off and save supernatant. (This is the soluble fraction).
#Draw off and save supernatant. (This is the soluble fraction).
-
#Resuspend pellet in 50 &mu;L [[Drummond:Solubility#Materials solubilization buffer]].
+
#Resuspend pellet in 100 &mu;L [[Drummond:Solubility#Materials|solubilization buffer]].
#Centrifuge at 10000 x ''g'' for 20 mins at 4&deg;C.
#Centrifuge at 10000 x ''g'' for 20 mins at 4&deg;C.
#Draw off and save supernatant.  (This is the insoluble fraction).
#Draw off and save supernatant.  (This is the insoluble fraction).

Revision as of 13:16, 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, a bacterial protocol. Here, the organisms is assumed to be S. cerevisiae.)

Total protein:

  1. Grow a 6mL overnight 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°C.
  4. Resuspend in 100 μL denaturing lysis buffer + 2% SDS.
  5. 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.
  6. 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.
  7. Incubate cells with agitation for 1 hr at room temperature.
  8. Centrifuge lysate at 10000 x g for 30 mins at room temperature.
    • 10 mins might be enough.
  9. Draw off and save supernatant. (This is the total protein fraction.)

Soluble and insoluble fractions:

  1. Take a 2mL aliquot of culture and move to 2 mL centrifuge tube
  2. Pellet cells by spinning at 4000 x g for 15 mins at 4°C.
  3. Resuspend in 100 μL native buffer
  4. Lyse cells
    • Use glass-bead protocol
  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. Draw off and save supernatant. (This is the soluble fraction).
  9. Resuspend pellet in 100 μL solubilization buffer.
  10. Centrifuge at 10000 x g for 20 mins at 4°C.
  11. Draw off and save supernatant. (This is the insoluble fraction).

Materials

Denaturing lysis buffer

Native lysis buffer

1% v/v Triton X-100

Solubilization buffer

2% v/v SDS 1% v/v Triton X-100

Links to protocols

Knight:Protein solubility

References

  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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