Drummond:Solubility

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Principle: protocol)
(protocol improvements)
Line 8: Line 8:
==Protocol==
==Protocol==
-
(Adapted from [[Knight:Protein solubility]].)
+
(Adapted from [[Knight:Protein solubility]], a bacterial protocol.  Here, the organisms asssumed to be <i>S. cerevisiae</i>.)
-
#Grow a 6mL culture.
+
Total protein:
 +
#Grow a 6mL overnight culture.
#Take 2mL of culture and move to 2mL centrifuge tube.
#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.
-
##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.
+
#Resuspend in 100 &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.''
-
##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.''
-
##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.''
+
#Centrifuge lysate at 10000 x ''g'' for 30 mins at room temperature.
-
##*''Kathleen suggests just lysing by heating at 90&deg;C for 10 mins but this may require the presence of SDS loading buffer?''
+
#*''10 mins might be enough.''
-
##Centrifuge lysate at 10000 x ''g'' for 30 mins at room temperature.
+
#Draw off and save supernatant.  (This is the total protein fraction.)
-
##*''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).
+
 +
Soluble and insoluble fractions:
 +
#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.
 +
#Resuspend in 100 &mu;L of [[#Materials/.
 +
#Lyse
 +
#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.''
 +
#Draw off and save supernatant. (This is the soluble fraction).
 +
#Resuspend pellet in 50 &mu;L [[Drummond:Solubility#Materials solubilization buffer]].
 +
#Centrifuge at 10000 x ''g'' for 20 mins at 4&deg;C.
 +
#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==
==Links to protocols==
[[Knight:Protein solubility]]
[[Knight:Protein solubility]]
==References==
==References==
 +
<biblio>
 +
#Marblestone-ProtSci-2006 pmid=16322573
 +
</biblio>

Revision as of 13:13, 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 asssumed 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 of [[#Materials/.
  4. Lyse
  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 50 μL Drummond:Solubility#Materials 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]
Personal tools