User:Linh N Le/Notebook/2009/06/23: Difference between revisions

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* Try Kinesin?  
* Try Kinesin?  
* Clean up lab
* Clean up lab
12-6


==BRB80==
==BRB80==
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***1 mM EGTA
***1 mM EGTA
***7 mMTaxol
***7 mMTaxol
**Andy mentions that he thinks these buffers are what Block used to get kinesin to stick to beads. It not be what we want, but if the whole "soup" (Beads + kinesin + buffer) is added to the microtubes mix and it doesnt mess things up, this stuff should be fine.
*[http://www.stanford.edu/group/blocklab/Valentine%20Fordyce%20et%20al%20NCB%202006.pdf Valentine, Fordyce, Block]
*[http://www.stanford.edu/group/blocklab/Valentine%20Fordyce%20et%20al%20NCB%202006.pdf Valentine, Fordyce, Block]
**polymerization buffer for brain tubulin  
**polymerization buffer for brain tubulin  
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***0.9 mM GTP  
***0.9 mM GTP  
***10% v/v DMSO
***10% v/v DMSO
 
**stablilzation buffer
***80 mM PIPES at pH 6.9
***1.7 mM EGTA
***5.5 mM MgCl2
***1.2 mM GTP
***8.2 mM sodium azide
***20 μM taxol
***10% v/v DMSO)
**Assay buffer based on PEM80
***80 mM PIPES at pH 6.9
***1 mM EGTA
***4 mM MgCl2


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Revision as of 16:30, 24 June 2009

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To Do

  • Koch/Atlas group meeting at 3
  • Try out new buffer we made yesterday?
  • Try Kinesin?
  • Clean up lab

12-6

BRB80

  • Since there are so many ways to make this buffer, Koch wanted me to try to find different famous people's recipes for the buffer
    • People to look for, Block and Vale
  • What we use for Cytoskeleton is called PEM and while PEM and BRB80 can be the same, they do not have to be
    • I did searches on both PEM and BRB80 and the following recipes are also reported by what they are called
  • Special Note: All PDF's linked here are from the personal website of the authors and are free access

Recipes

  • Hancock (Used by us as well)
    • 80 mM PIPES pH 6.85 (we use ph6.9)
    • 1 mM MgCl2
    • 1 mM EGTA
  • T.Salmon Labs
    • PM Buffer
      • 100 mM PIPES, pH 6.9
      • 2 mM EGTA
      • 1 mM Mg2SO4
  • Goldman Lab (Northwestern University)
    • 100 mM Pipes,pH 6.9
    • 1 mM MgCl2
    • 1 mM EGTA
  • Cold Spring Harbor Labs
    • PEM Buffer
      • 100 mM PIPES (pH 6.95)
      • 2 mM EGTA
      • 1 mM MgSO4
  • Cytoskeleton
    • PEM (General Tubulin Buffer)
      • 80 mM Na-PIPES pH 6.9
      • 1 mM MgCl2
      • 1 mM EGTA
  • Mitchison Lab (Harvard)
    • Brinkley Buffer 1980 (BRB80)
      • 80mM PIPES pH 6.8
      • 1mM MgCl2
      • 1mM EGTA
    • "Cytoskeleton Buffer"
      • 10mM MES pH 6.1
      • 138mM KCl
      • 3mM MgCl
      • 2mM EGTA
    • Notes: BRB80 is good for microtubules and 'cytoskeleton buffer' is good for both actin filaments and microtubules
  • Sigma Aldrich
    • PEM Buffer:
      • 0.1M PIPES (Product No. P8203)
      • 5 mM EGTA (Product No. E4378)
      • 2mM MgCl2 · 6H2O (Product No. M0250)
      • Bring to pH 6.8, using NaOH solution
  • Bayer College of Medicine
    • PEM Buffer:
      • 80mMPotassium PIPES, pH 6.8
      • 5 mM EGTA, pH 7.0
      • 2 mM MgCl2 ( final Concentration: 2 mM)
  • Yu-li Wang from Carnegie Mellon This is a pdf of how they do Rhodamine Tubulin
    • PEM buffer:
      • 0.1 M PIPES
      • 1.0 mM EGTA
      • 0.5 mM MgCl2
      • pH 6.9
  • Tedeschi of Italy
    • PEM buffer
      • 85 mM Pipes, pH 6.94
      • 10 mM EGTA
      • 1 mM MgCl2
  • Steve Gross of U Minnesota with Steve Block
    • 80 mM Pipes
    • 1 mM EGTA
    • 4 mM MgCl2
  • Stefan Diez (Germany)
    • BRB80 buffer
      • 80 mM potassium Pipes
      • 1 mM EGTA
      • 1 mM MgCl2
      • pH 6.9
  • Mathew Lang, Steve Block first author
  • Also used in Rosenfield, Block Paper
  • Also used in Guydosh Block Paper with 10μM of Taxol
    • Un-named buffer
      • 80 mM Pipes (pH6.9)
      • 50 mM potassium acetate
      • 4 mM MgCl2
      • 2 mM DTT
      • 1 mM EGTA
      • 7 mMTaxol
    • Andy mentions that he thinks these buffers are what Block used to get kinesin to stick to beads. It not be what we want, but if the whole "soup" (Beads + kinesin + buffer) is added to the microtubes mix and it doesnt mess things up, this stuff should be fine.
  • Valentine, Fordyce, Block
    • polymerization buffer for brain tubulin
      • 62 mM PIPES at pH 6.9
      • 0.8 mM EGTA
      • 3.7 mM MgCl2
      • 0.9 mM GTP
      • 10% v/v DMSO
    • stablilzation buffer
      • 80 mM PIPES at pH 6.9
      • 1.7 mM EGTA
      • 5.5 mM MgCl2
      • 1.2 mM GTP
      • 8.2 mM sodium azide
      • 20 μM taxol
      • 10% v/v DMSO)
    • Assay buffer based on PEM80
      • 80 mM PIPES at pH 6.9
      • 1 mM EGTA
      • 4 mM MgCl2
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