It's just a curiosity that could also help me in my troubleshooting: does Laemmli buffer behave "normally" without the lysate? Like, what happens if I load in the well 20 μL of Laemmli instead of 20 μL of Laemmli and sample? Does it show anyway one neat band during and after gel running?
I'm asking it because I can't obtain a neat band after SDS-PAGE but the protein standard column is fine, and I want to understand where is the problem
It contains buffer salt with an adjusted pH (Tris), glycerol for density, bromophenol blue as dye front to track the running time, SDS to serve negative charge and improve denaturation and the linearization of the proteins, and a reducing agent to break the disulfide bonds thereby unfolding the proteins. Heat treatment is also applied typically for denaturation within these reagents. Without most of these agents, you would go native PAGE but your running buffer and gel of course should be ok for this application. Protein ladders are ready to use it contains a dye and linear protein mixture, therefore they can give neat bands even they did not pretreat with Laemmli sample buffer. I do not know this one is the proper answer to your question but I hope that it would be useful for your research and troubleshoot.
Emir
Maybe your running buffer is not the correct pH or composition? I have had weird running bands in the past, but the protein ladder was fine, and I think it was because of the running buffer (but I cannot know for sure). Hope that helps! For troubleshooting I would not recommend adding laemli buffer slone because I don't think it will tell you much.
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