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Sauer:bis-Tris SDS-PAGE, the very best: Difference between revisions
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Sauer:bis-Tris SDS-PAGE, the very best (view source)
Revision as of 07:44, 26 August 2005
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Submitted by | Submitted by [[Sean Moore]] | ||
Based on work done by Tim Updyke and Sheldon Engelhorn for the Invitrogen Corporation (they bought Novex who developed it) and detailed in U.S. Patent 6,162,338. These gels are sold by Invitrogen as NuPAGE MES-Tris gels. | Based on work done by Tim Updyke and Sheldon Engelhorn for the [http://www.invitrogen.com/ Invitrogen Corporation] (they bought Novex who developed it) and detailed in U.S. Patent [http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=/netahtml/srchnum.htm&r=1&f=G&l=50&s1=6,162,338.WKU.&OS=PN/6,162,338&RS=PN/6,162,338 6,162,338]. These gels are similar to those sold by Invitrogen as NuPAGE MES-Tris gels. | ||
==Background== | |||
The pH of the separating gel in “standard” SDS-PAGE (a.k.a. Laemmli buffer system) is roughly 8-9 which is conducive to the deamination and alkylation of proteins, as well as reoxidation of reduced cysteines during electrophoresis. What this means is that your protein will form disulfide crosslinks during the stacking event because the protein migrates into the gel away from the reducing reagent in the sample buffer, and gets focused to a high concentration. Acrylamide gels cast in alkaline buffers are also unstable during long term storage, breaking down to acrylic acid after 1 to 2 months resulting in loss of pore size, poor resolution, and modified proteins. | The pH of the separating gel in “standard” SDS-PAGE (a.k.a. Laemmli buffer system) is roughly 8-9 which is conducive to the deamination and alkylation of proteins, as well as reoxidation of reduced cysteines during electrophoresis. What this means is that your protein will form disulfide crosslinks during the stacking event because the protein migrates into the gel away from the reducing reagent in the sample buffer, and gets focused to a high concentration. Acrylamide gels cast in alkaline buffers are also unstable during long term storage, breaking down to acrylic acid after 1 to 2 months resulting in loss of pore size, poor resolution, and modified proteins. | ||
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'''Sample bis-Tris MES-Tris Gels''' The first gel is of purified proteins. The second gel is of total ''E. coli'' lysate with and without an induced protein. Note the broad separation range of the molecular weight markers. | '''Sample bis-Tris MES-Tris Gels''' The first gel is of purified proteins. The second gel is of total ''E. coli'' lysate with and without an induced protein. Note the broad separation range of the molecular weight markers. | ||
==Materials== | |||
===acrylamide=== | |||
30% acrylamide | 30% acrylamide | ||
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Alternately, you can use to 30/0.8 mix, just make the gel 12-15% final. | Alternately, you can use to 30/0.8 mix, just make the gel 12-15% final. | ||
===<u>5X</u> low-MW running buffer=== | |||
Use for separating small proteins 2-50 kDa. | Use for separating small proteins 2-50 kDa. | ||
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0.5% SDS | 0.5% SDS | ||
No need to pH. | No need to pH. | ||
add sodium bisulf<u>ite</u> to 5 mM (add fresh before run)from a 1M stock. It's stinky. | add sodium bisulf<u>ite</u> to 5 mM (add fresh before run)from a 1M stock. It's stinky. | ||
===<u>5X</u> high-MW running buffer=== | |||
use for separating proteins >20 kDa. | use for separating proteins >20 kDa. | ||
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add sodium bisulf<u>ite</u> to 5 mM (add fresh before run)from a 1M stock. It's stinky. | add sodium bisulf<u>ite</u> to 5 mM (add fresh before run)from a 1M stock. It's stinky. | ||
===200X running buffer reducing agent=== | |||
1 M sodium bisulfite | 1 M sodium bisulfite | ||
add to running buffer at 5 mM final concentration | add to running buffer at 5 mM final concentration | ||
===3.5X gel buffer=== | |||
1.25 M bis-Tris (pH 6.5-6.8 with HCl) | 1.25 M bis-Tris (pH 6.5-6.8 with HCl) | ||
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note bis-Tris is Bis(2-hydroxyethyl) aminotris (hydroxymethyl) methane (e.g. Sigma catalog# B 7535). | note bis-Tris is Bis(2-hydroxyethyl) aminotris (hydroxymethyl) methane (e.g. Sigma catalog# B 7535). | ||
===sample buffer=== | |||
I routinely use the "standard" Laemmli 3X buffer. I think it does a better job reducing samples because of the higher pH. | I routinely use the "standard" Laemmli 3X buffer. I think it does a better job reducing samples because of the higher pH. | ||
==Casting and running gels== | |||
===Resolving:=== | |||
Mix: 1/3.5 vol. of 3.5X bis-Tris gel buffer, acrylamide to 8% (30:2.0) or 12-15% (30:0.8), and water to final volume. I make 3.75 mLs for each Bio-Rad Protean gel, and use 3.5 mLs per gel. | Mix: 1/3.5 vol. of 3.5X bis-Tris gel buffer, acrylamide to 8% (30:2.0) or 12-15% (30:0.8), and water to final volume. I make 3.75 mLs for each Bio-Rad Protean gel, and use 3.5 mLs per gel. | ||
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Drain the butanol, rinse with water, wick the water from between the plates with a shred of filter paper. | Drain the butanol, rinse with water, wick the water from between the plates with a shred of filter paper. | ||
===Stacking:=== | |||
1X bis-Tris gel buffer, acrylamide solution to 4%, water. | 1X bis-Tris gel buffer, acrylamide solution to 4%, water. | ||
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Rinse with water to remove unpolymerized acrylamaide. Remove comb. | Rinse with water to remove unpolymerized acrylamaide. Remove comb. | ||
===Running=== | |||
Fill both upper and lower buffer chambers with either MES-Tris or MOPS-Tris buffer. | Fill both upper and lower buffer chambers with either MES-Tris or MOPS-Tris buffer. | ||
Run at 150V constant. The Bromophenol blue runs around 3-5 kDa. | Run at 150V constant. The Bromophenol blue runs around 3-5 kDa. | ||
