When it comes to choosing a molecular weight marker to run on your SDS-PGE gels, there are a lot of options out there. How do you know which one is right for you? Read on for tips on what to consider when choosing a standard for your protein gels.
Before you go about selecting a molecular weight marker, ask yourself a few basic questions about your experiment.
First, what size proteins are you running?
Molecular weight markers come in different size ranges to accommodate a wide range of target protein sizes. A standard marker will have bands representing around 30 kDa to around 150 kDa, although the effective resolution can get a little vague at the ends of the spectrum. You can also purchase markers specific for very small proteins, or very large proteins. These ladders will give enable you to more accurately gauge the size of your protein. If you’re looking at a range of protein sizes, you can use an extended range markers, which will give you more options at the extreme ends of the protein size spectrum.
Second, what downstream applications will you be using your gel for?
Will you be Commassie-staining your gel, or transferring it to do a Western blot? If you will be doing a Western, you may want to consider using an IgG-labeled marker (such as the Invitrogen MagicMark) that will be stained with your secondary antibody during the blotting process. This eliminates the need for overlaying your membrane on the film (or image) to figure out what protein bands match up to what sizes. For fluorescence, you may want to try the Biorad Western C.
If you’ll be doing a Coommassie stain, you can get away with using a completely unstained ladder, as it will pick up the Coommassie dye just like any other collection of proteins. However, most people use a pre-stained ladder, regardless of whether they’ll be staining or blotting later, because it can help you monitor the run time of your gel. As we discussed earlier, a pre-stained ladder (like Novex Sharp or Biorad’s Precision Plus Dual Colour) can also help you monitor your transfer conditions. (Note that we have suggested these specific products only because they work in our hands, but there are plenty of others available!)
Finally, what information do you need your ladder to tell you?
Will your blot/gel be quantitative? Do you need to be able to accurately calculate the protein size? Or do you just need a general idea of where the protein is running? For more accurate protein measurement, be sure to select a molecular weight standard with the appropriate size range, as mentioned above. If you want to be able to monitor transfer of the proteins to a membrane, choose a pre-stained ladder.
For more information about molecular weight markers, SDS-PAGE and western blotting, check out this video by Agrisera.
You might think Northern Blots are an old-fashioned technique. However, qRT-PCR is prone to false positives and negatives, and reviewers may require Northern Blot confirmation of your qRT-PCR results. So sometimes Northern Blots are a necessary evil.
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