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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 13:20, 19 August 2005
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'''SDS-PAGE, buffered with bis-Tris''' | <large><u>'''SDS-PAGE, buffered with bis-Tris'''</u></large> | ||
Submitted by Sean | Submitted by Sean | ||
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<u>'''Materials'''</u> | <u>'''Materials'''</u> | ||
'''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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add sodium bisulf | 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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No Need to pH. | No Need to pH. | ||
add sodium bisulf | 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. | ||
''' | '''<u>Casting and running gels</u>''' | ||
'''Resolving:''' | '''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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Rinse with water to remove unpolymerized acrylamaide. Remove comb. | Rinse with water to remove unpolymerized acrylamaide. Remove comb. | ||
'''Running''' | '''Running''' | ||
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. | ||
