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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 09:19, 19 December 2019
, 19 December 2019→sample buffer
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===sample buffer=== | ===sample buffer=== | ||
You can use any of the the common Laemmli-like buffers. | |||
<b>Stock reagents</b> | |||
Make separate 200 mM Tris-Cl, pH 6.8 | |||
Make separate 50% glycerol | |||
Make separate 10% SDS | |||
Make separate 0.1% bromophenol blue (may look a bit orange because of low pH, that's OK) | |||
Aliquot some 2-mercaptoethanol into a microfuge tube, keep on-hand (100% is ~14.3 M). | |||
Mix the ingredients to some preferred "X", 2X is common: | |||
<b>2X</b> | |||
100 mM Tris-Cl, pH 6.8 | |||
20% glycerol | |||
2% SDS | |||
0.01% bromophenol blue | |||
store this concentrate and add 2-mercaptoethanol to 5% (~700 mM) to an aliquot within a few days of use. | |||
Commercial 2X - 4X versions are found with different formulations, but most have the low pH Tris ranging from ~32 to 70 mM in the 1X, which provides Cl to aid in the stacking event. | |||
The Laemmli original 1X is (Laemmli, 1970): | |||
62.5 mM Tris-Cl, pH 6.8 | |||
2% SDS | |||
10% glycerol | |||
0.0006% bromophenol blue | |||
5% 2-mercaptoethanol | |||
==Casting and running gels== | ==Casting and running gels== | ||
