I am trying to run an SDS-PAGE gel to find a apolipoproteinB-100 which is 550kDa. I found a few papers that referenced Laemmli's SDS-PAGE protocol and it says that the protein sample, the sample buffer, and the protein MW standard must be included in each well. Because both the sample buffer and standard are dyed are both necessary? Could the protein standard be added to a blank well and the sample buffer added to the rest?
Thank you all so much!
Dear Courtney
First of all, 550KDa is a very high mW and is not easy to be resolved in SDS.page. You can try using 3-7% tris-acetate gels.
https://www.thermofisher.com/content/dam/LifeTech/global/Forms/PDF/protein-gel-electrophoresis-technical-handbook.pdf
Regarding the proteins standard. As you suggest, you can clearly add the protein marker in a separate clear well and you do not need to mix it with the samples, also because in case that the bands of the marker run at the same MW of the sample probably you will not detect them well.
Probably in the articles, especially if are old articles, suggest to use for both marker and sample the SDS-page running buffer and probably they also suggest that thanks to the presence of bromophenol blue as tracking agent, the Standard SDS sample buffer could be used to track the gel running.
However while in the past the bromophenol blue was essential to follow the gel run, recently the increasing availability of pre-stained protein marker (eg Novex Sharp prestained cod LC5800) that can be visualized also during the run and can help you stop the gel at the best moment.
good luck
Manuele
Hi Broady,
I agreed with Manuele's suggestion that SDS-PAGE won't give you good resolution for protein of that high MW.Also in terms of your sample protein, load protein marker MW on a separate well (MW protein marker+4x-SDS dye) and on another separate well(your sample protein +4x-SDS dye).
I hope this will help .
Hello Courtney,
no, sample and ladder must be in seperate wells. How would you know which band belongs to your sample and which to your marker?
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