Drummond:Lysis

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==Chemical lysis==
==Chemical lysis==
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Myristyl sulfobetaine
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The major active ingredient in Y-Per lysis reagent is apparently 10 mg/mL SB3-14 (myristyl sulfobetaine)<cite>ZannaJChr07</cite>, also known as
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3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB14) <cite>ZannaJChr07</cite>, available from Sigma as [http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/T7763 SB3-14].
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3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate or SB14, available from Sigma as [http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/T7763 SB3-14].  Y-Per is a protein-free reagent and SB3-14 is the only detergent <cite>ZannaJChro07</cite>.
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For assessing soluble versus insoluble fractions of proteins, chemical lysis of membranes may not be ideal.  The problem is that if detergents liberate proteins from the membrane efficiently, then we can expect them to solubilize some insoluble proteins, and reduce signal.  The appeal of mechanical lysis is that it is not expected to alter the solubility of proteins.
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Idea: Mechanically disrupt, spin, and extract supernatant.  Spin down to clarify.  Draw off supernatant as soluble fraction and save pellet.  Wash beads with 100 &mu;L solubilization buffer, vortex, and draw off as much material as possible (liquid + pellet).  Add to pellet remaining after soluble fraction removal.  Vortex to resuspend and incubate 5 min at room temperature.  Spin down to clarify.  Draw off supernatant as insoluble fraction.
==References==
==References==

Revision as of 13:47, 7 July 2007

SUME Buffer (1% SDS, 8M Urea, 10mM MOPS, pH 6.8, 10mM EDTA) To make 100ml:

   * 1.0ml 1M MOPS, pH6.8 stock solution
   * 1.0g SDS, or 10ml 10% SDS stock solution
   * 48.05g Urea
   * 2.0ml 0.5M EDTA stock solution

This is used for lysing yeast at pH6.8. For pH8 lyses, use the variation of SUME with Tris buffer known as SUTE.

from Yeast Protocols

Chemical lysis

The major active ingredient in Y-Per lysis reagent is apparently 10 mg/mL SB3-14 (myristyl sulfobetaine)[1], also known as 3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate or SB14, available from Sigma as SB3-14. Y-Per is a protein-free reagent and SB3-14 is the only detergent [2].

For assessing soluble versus insoluble fractions of proteins, chemical lysis of membranes may not be ideal. The problem is that if detergents liberate proteins from the membrane efficiently, then we can expect them to solubilize some insoluble proteins, and reduce signal. The appeal of mechanical lysis is that it is not expected to alter the solubility of proteins.

Idea: Mechanically disrupt, spin, and extract supernatant. Spin down to clarify. Draw off supernatant as soluble fraction and save pellet. Wash beads with 100 μL solubilization buffer, vortex, and draw off as much material as possible (liquid + pellet). Add to pellet remaining after soluble fraction removal. Vortex to resuspend and incubate 5 min at room temperature. Spin down to clarify. Draw off supernatant as insoluble fraction.

References

  1. Zanna L and Haeuw JF. . pmid:16978932. PubMed HubMed [ZannaJChr07]
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