Gel ladders - tips and tricks for using molecular weight markers

Gel “ladders,” or “molecular weight markers” are simply mixes of molecules (proteins, DNA, or RNA) of known sizes which you can run alongside your samples in gel electrophoresis for comparison. In electrophoresis, we use electricity to send molecules swimming through a gel mesh (made of agarose or polyacrylamide). The bigger* molecules get caught up in the mesh more so they swim slower. And when we turn off the power box, they’ll be higher up in the gel than smaller ones. But how much higher? We can stain the gel with a dye like Coomassie blue which will dye proteins or ethidium bromide or a SYBR fluorescent stain ton dye nucleic acids and show us their location. But we can’t even estimate size without a comparison to something of known size, and this is where the ladder comes in. Here are some tips for choosing using them and some cool “bonuses” you can use them for - like did you know prestained protein ladders are often fluorescent too?
* (well, usually more accurately, longer since the molecules are typically denatured (unfolded) first)
blog form: bit.ly/ladder_...
First off, you need to choose your ladder.
Choose a ladder that encompasses the entire range of product sizes you expect are in your sample (you want each of your bands to be between 2 ladder bands in order to estimate size).
Unless you’re working with massive proteins or small peptides, protein ladders are typically more universal. I usually use Precision Plus Protein Standards from BIO-RAD which encompass 10-250 kDa (kiloDaltons, a measure of molecular weight. More on kDa here: blog: bit.ly/protein... ; RUclips: • Protein concentrations...
But a rough estimate is that the average amino acid (protein letter) is about 0.1 kDa, so divide a protein length by 10 to get a kDa estimate - e.g a 500 amino acid protein would have a molecular weight of ~50 kDa)
If you are working with something big or tiny, they have special ladders you can get for that.
For nucleic acids, the range of sizes you might want to visualize can vary a lot a lot. You might, for example, want to visualize microRNAs (~20 nucleotides long) or big linearized plasmids (in the thousands of nucleotides). So you need to choose the ladder that fits your needs.
For general cloning purposes, a 1 kb ladder typically does the trick. Note on the naming: kb stands for kilo base pairs (1000 base pairs - we talk in terms of base pairs rather than bases (individual letters) because the DNA is double-stranded). These ladders are often named in terms of the size of the increment between the bands. So most of the bands in the 1 kb ladder are 1 kb apart - but some of the smaller ones are closer together. There are also 100 bp and 10 bp ladders for dealing with small stuff.
No matter what type of molecule, there can be slight differences in how molecules run compared to the standards because your molecules will have slightly different compositions. Therefore, your band might be slightly higher or lower than where you’d expect based on the ladder but don’t freak out! If you have a positive control of the exact band you’re looking for that can be very helpful. For nucleic acids, even if you don’t have the exact molecule as a control, you can order oligos (short pieces of DNA or RNA) of precise sizes not present in the ladder to serve as custom size markers - this can be helpful if you’re trying to PAGE purify or something. more on that here: blog: bit.ly/gel_ext... ; RUclips: • Gel extraction with th...
Also be forewarned that the ladders will run differently depending on the gel type and percentage and the buffer type. So your gel might not look quite like the picture on that free magnet you get as SWAG. Often, especially for the bigger bands, they scrunch together and can be hard to tell apart. This is a place where positive controls can come in handy.
Sometimes you also have a problem with the really low molecular weight bands in that they’re too faint (because the molecules are smaller there’s less for the dye to bind to so the same number of the smaller molecules appears fainter). Or they might even run out of the gel.
This could leave you guessing which band is which. Thankfully the ladder makers typically include more of a few of the molecules as “reference bands” - in the case of Precision Plus ladders, these are the 25, 50, and 75 kDa bands - which show up stronger and allow you to orient things. This orientation is even easier if you use a multi-colored prestained ladder, where the bands are different colors.
Typically, because they’re more expensive, we reserve the prestained ladders for things like western blots, where we want to be able to visualize that the bands get transferred out of the gel and onto the membrane, and so that, where applicable, we know where to cut the membrane. It also allows us to monitor the run progression.
Finished in comments

[@abdelfattahalanati5821]

You are such a hard worker, I like your videos, keep going 👏

[@thebumblingbiochemist]

Thanks!

[@thebumblingbiochemist]

When I’m doing western blots, I typically use an all blue prestained ladder. It doesn’t have the benefit of multiple colors, but it does have the benefit that all of the bands can be detected with the same wavelength (635 nm) which is useful for when you’re using fluorescent antibodies for band detection - you’re able to visualize the ladder easily.
Some ladders come premixed with loading buffer and others you have to mix yourself. For the ones you mix yourself, it can be helpful to prepare more than you need and store aliquots. This saves you time in the future and prevents multiple freeze-thaws.
Might seem like a pain to have to mix in the dye but it allows you to more finely control the concentration you load, which is crucial for making sure you load a similar quantity/concentration of ladder and sample. For example, DNA ladders are often at 1 μg/μL of DNA and loading dye is often at 6X concentration. If you wanted to load 1 μg in a 6 μL volume, you’d mix 1 μL ladder with 4 μL water (or a buffer like TE) and 1 μL loading dye.
Depending on how the concentration of your sample, that might be too strong of a signal (you don’t want it to drown out your bands and/or make it hard to autofocus detector). So you can load less volume or do a greater dilution (e.g. maybe dilute 0.5 μL with 4.5 μL water/buffer). It can be hard to accurately pipet that small of volumes, but you can create a stock solution with many reactions-worth, and freeze aliquots of it. Alternatively, you can dilute a larger amount, but then only mix 5 μL-worth with the loading dye.
The ladders can degrade, and this makes it hard to tell the bands apart. Especially for the nucleic acid ladders, you want to avoid repeated freeze-thaws, so you can store aliquots of pre-mixed ladder.
much more on gel electrophoresis: bit.ly/agarosegelcompare
and gel smiling: bit.ly/gel_smile
more on western blots: bit.ly/westernblotworkflow
more on radiolabeling: bit.ly/radiolabelings
BIO-RAD’s Little Book of Standards: www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_2414.pdf
   
    
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com