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

From OpenWetWare
< User:Linh N Le‎ | Notebook‎ | 2009‎ | 06
Jump to navigationJump to search
(22 intermediate revisions by the same user not shown)
Line 11: Line 11:
* Try Kinesin?  
* Try Kinesin?  
* Clean up lab
* Clean up lab
12-6


==BRB80==
==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
*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
**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===
===Recipes===
*Hancock (Used by us as well)
*[http://www.proweb.org/kinesin/Methods/motility.html Hancock (Used by us as well)]
**80 mM PIPES pH 6.85 (we use ph6.9)
**80 mM PIPES pH 6.85 (we use ph6.9)
**1 mM MgCl2
**1 mM MgCl2
**1 mM EGTA
**1 mM EGTA
*T.Salmon Labs
*[http://www.proweb.org/kinesin/Methods/MT_assembly.html T.Salmon Labs]
**PM Buffer
**PM Buffer
***100 mM PIPES, pH 6.9  
***100 mM PIPES, pH 6.9  
***2 mM EGTA
***2 mM EGTA
***1 mM Mg2SO4
***1 mM Mg2SO4
*Goldman Lab (Northwestern University)
*[http://www.goldmanlab.northwestern.edu/protocols.htm Goldman Lab (Northwestern University)]
**100 mM Pipes,pH 6.9  
**100 mM Pipes,pH 6.9  
**1 mM MgCl2
**1 mM MgCl2
**1 mM EGTA
**1 mM EGTA
 
*[http://cshprotocols.cshlp.org/cgi/content/full/2006/20/pdb.rec10559?text_only=true Cold Spring Harbor Labs]
**PEM Buffer
***100 mM PIPES (pH 6.95)
***2 mM EGTA
***1 mM MgSO4
*[http://www.cytoskeleton.com/products/buffers/bst01.html Cytoskeleton]
**PEM (General Tubulin Buffer)
***80 mM Na-PIPES pH 6.9
***1 mM MgCl2
***1 mM EGTA
*[http://mitchison.med.harvard.edu/protocols/gen1.html 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
*[http://www.sigmaaldrich.com/life-science/cell-biology/antibodies/antibodies-application/protocols/immunofluorescence.html 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
*[http://www.bcm.edu/microscopy/?PMID=2855 Bayer College of Medicine]
**PEM Buffer:
***80mMPotassium PIPES, pH 6.8
***5 mM EGTA, pH 7.0
***2 mM MgCl2 ( final Concentration: 2 mM)
*[http://www.ece.cmu.edu/~yuliwang/Methods/Materials/Fluorescent%20Labelilng/RhodamineTubulin.pdf 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
*[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T0G-4JKHMBD-4&_user=1550512&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=937840362&_rerunOrigin=google&_acct=C000053660&_version=1&_urlVersion=0&_userid=1550512&md5=a51e4c2c3d082850d44f8d0824033a88 Tedeschi of Italy]
**PEM buffer
***85 mM Pipes, pH 6.94
***10 mM EGTA
***1 mM MgCl2
*[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B94RW-4TX1P02-8&_user=1550512&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000053660&_version=1&_urlVersion=0&_userid=1550512&md5=a4a0f0dc1f0a705362349f6ef05e3489 Steve Gross of U Minnesota with Steve Block]
**80 mM Pipes
**1 mM EGTA
**4 mM MgCl2
*[http://pubs.acs.org/doi/full/10.1021/nl060922l?cookieSet=1 Stefan Diez (Germany)]
**BRB80 buffer
***80 mM potassium Pipes
***1 mM EGTA
***1 mM MgCl2
***pH 6.9
*[http://www.stanford.edu/group/blocklab/Block%20et%20al%20PNAS%202003.pdf Mathew Lang, Steve Block first author]
*Also used in [http://www.stanford.edu/group/blocklab/Rosenfeld%202003.pdf Rosenfield, Block Paper]
*Also used in [http://www.stanford.edu/group/blocklab/Guydosh%20and%20Block.pdf 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.
*[http://www.stanford.edu/group/blocklab/Valentine%20Fordyce%20et%20al%20NCB%202006.pdf 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


<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Revision as of 16:30, 24 June 2009

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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
Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Linh_N_Le/Notebook/2009/06/23&oldid=318794"