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== | ||
*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 | ||
*[www.goldmanlab.northwestern.edu/protocols. | *[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 | ||
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*[http://cshprotocols.cshlp.org/cgi/content/full/2006/20/pdb.rec10559?text_only=true Cold Spring Harbor Labs] | *[http://cshprotocols.cshlp.org/cgi/content/full/2006/20/pdb.rec10559?text_only=true Cold Spring Harbor Labs] | ||
**PEM Buffer | **PEM Buffer | ||
*** | ***100 mM PIPES (pH 6.95) | ||
***2 mM EGTA | ***2 mM EGTA | ||
***1 mM MgSO4 | ***1 mM MgSO4 | ||
*Cytoskeleton | *[http://www.cytoskeleton.com/products/buffers/bst01.html Cytoskeleton] | ||
**PEM (General Tubulin Buffer) | **PEM (General Tubulin Buffer) | ||
***80 mM Na-PIPES pH 6.9 | ***80 mM Na-PIPES pH 6.9 | ||
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***2mM EGTA | ***2mM EGTA | ||
**Notes: BRB80 is good for microtubules and 'cytoskeleton buffer' is good for both actin filaments and microtubules | **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 | |||
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Revision as of 16:30, 24 June 2009
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To Do
12-6 BRB80
Recipes
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