369
edits
Sauer:bis-Tris SDS-PAGE, the very best: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Sauer:bis-Tris SDS-PAGE, the very best (view source)
Revision as of 09:22, 14 June 2022
, 14 June 2022→sample buffer
| Line 72: | Line 72: | ||
===sample buffer=== | ===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. 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. | 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. Their recipe tends to cause the SDS to crash out of solution, so it has to be reheated each time before aliquoting into tubes. We also have found that having Cl- in the sample helps stacking (sharpness), so a simple fix is to mix Laemmli buffer and the phosphate buffer 1:1. | ||
<b>Stock reagents</b> | <b>Stock reagents</b> | ||
