Sauer:bis-Tris SDS-PAGE, the very best: Difference between revisions

Sauer:bis-Tris SDS-PAGE, the very best (view source)

386 bytes added , 14 June 2022

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 ===sample buffer===
-You can use any of the the common Laemmli-like buffers. Many people over-cook their proteins and cause brakdown, probably because Laemmli was solubilizing a protein pellet. A short heating of ~1 min at ~80-100 C is more than sufficient and only has to be done once to fully denature the proteins and coat them. Using a lower temp, like 40-60 C to thaw frozen aliquots helps to prevent ruining the samples.
+You can use any of the the common Laemmli-like buffers. Many people over-cook their proteins and cause brakdown, probably because Laemmli was solubilizing a protein pellet. A short heating of ~1 min at ~80-100 C is more than sufficient and only has to be done once to fully denature the proteins and coat them. Using a lower temp, like 40-60 C to thaw frozen aliquots helps to prevent ruining the samples. We can also used a phosphate-based buffer if you need to cook your samples longer (it controls the pH to prevent acidic proteolysis); it's based on this reference: Cannon-Carlson, S., Tang, J. (1997) "Modification of the Laemmli Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis Procedure to Eliminate Artifacts on Reducing and Nonreducing Gels" Anal. Biochem., 246:146–148.
 <b>Stock reagents</b>