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Extraction of membrane proteins Protocols [View Printable]
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R Bishop
Group: Admin
Posts: 304
Joined: Jan 17, 2006
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If you have experience with membrane protein extraction post your protocols and ideas here.
Rb |
.........................
"To those who would tear the world down: We will defeat you. To those who seek peace and security: We support you. And to all those who have wondered if America's beacon still burns as bright: Tonight, we proved once more that the true strength of our nation comes not from the might of our arms or the scale of our wealth, but from the enduring power of our ideals: democracy, liberty, opportunity and unyielding hope."
-- Barack Obama, Nov. 4, 2008
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| Posted Oct 13, 2006, 22:01 PM |
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R Bishop
Group: Admin
Posts: 304
Joined: Jan 17, 2006
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Extraction Buffer for Membrane Proteins
40mM Tris-base, 5M urea, 2M thiourea, 2% CHAPS, 2%SB 3-10, 5 mM TCEP or 10 mM DTT, Protease Inhibitors ( 5mM benzamidine, 5mM PMSF, 1mM N-ethylamide), pH 9.5
40mM Tris (pH 9.5)
1. Rinse cells with PBS 2X
2. 5mM EDTA lift cells, 10 min 37C
3. Wash cells 2x PBS
4. Resuspend total cells in 40mM Tris (pH 9.5)
5. Pass 10x thru a 22 gauge needle (30cc syringe), cells look whole but single
6. Extract membrane proteins 1/20 weight to volume in extraction buffer 3hrs or o/n 4C gentle shaking
Alternate
1. (5 M urea, 2 M thiourea, 2%
CHAPS, 40 mM Tris, 2% SB3-10, 0.2% Bio-lyte (5/8),
0.2% Bio-lyte (8/10), 2 mM TBP).
Here's a protocol I often follow
Rb
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.........................
"To those who would tear the world down: We will defeat you. To those who seek peace and security: We support you. And to all those who have wondered if America's beacon still burns as bright: Tonight, we proved once more that the true strength of our nation comes not from the might of our arms or the scale of our wealth, but from the enduring power of our ideals: democracy, liberty, opportunity and unyielding hope."
-- Barack Obama, Nov. 4, 2008
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| Posted Oct 13, 2006, 22:02 PM |
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R Bishop
Group: Admin
Posts: 304
Joined: Jan 17, 2006
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.........................
"To those who would tear the world down: We will defeat you. To those who seek peace and security: We support you. And to all those who have wondered if America's beacon still burns as bright: Tonight, we proved once more that the true strength of our nation comes not from the might of our arms or the scale of our wealth, but from the enduring power of our ideals: democracy, liberty, opportunity and unyielding hope."
-- Barack Obama, Nov. 4, 2008
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| Posted Oct 13, 2006, 22:04 PM |
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R Bishop
Group: Admin
Posts: 304
Joined: Jan 17, 2006
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Mem-PER Eukaryotic Membrane Protein Extraction Kit
Has anyone used this kit from Pierce to extract membrane proteins?
http://www.piercenet.com/products/browse.cfm?fldID=06010422
protocol available here
http://www.piercenet.com/Objects/View.cfm?type=File&ID=1312
Rb |
.........................
"To those who would tear the world down: We will defeat you. To those who seek peace and security: We support you. And to all those who have wondered if America's beacon still burns as bright: Tonight, we proved once more that the true strength of our nation comes not from the might of our arms or the scale of our wealth, but from the enduring power of our ideals: democracy, liberty, opportunity and unyielding hope."
-- Barack Obama, Nov. 4, 2008
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| Posted Oct 13, 2006, 22:06 PM |
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