There is so much information about proteins that might be quite confusing. However, this nutrient plays a vital role in our bodies, and the last thing you can do is amass as much information as possible. In this article, we will look at protein ladders. However, to help you quickly grasp what it is, we must look at electrophoresis. Electrophoresis, simply put, is an electrokinetic process, which separates the charged particles in a fluid through a field of electrical charge.
Therefore, it plays an essential role in separating the protein molecules, which is needed for a range of reasons. Now that you understand what this process is, let’s now focus on our main topic of discussion.
What are protein ladders?
Protein ladders are combinations of known molecular weight proteins that are used to construe the relative molecular sizes of the proteins that have been separated during electrophoresis. This process separates charged particles in fluids. What usually happens during this process is that molecular weight markers are generally in the outside lane of a gel. This is done to easily compare the results with the test samples in the middle lanes.
Each protein marker will make a certain distance as it interacts with the gel, after which a curve can be drawn. The molecular weight of these migrating protein samples will then be calculated by interpolation. Keep in mind that these molecular weight markers are mostly used as reference points. Therefore, they help scientists to identify the proteins of interest based on their sizing.
Relationship between MW marker and protein ladder
Let us continue to expand on this area if you didn’t get our first part. Well, MW marker or molecular weight marker are combinations of native proteins with well-characterized sizes and mobilities, which cannot, however, be construed in whole numbers. On the other hand, a protein ladder is basically combinations of labeled proteins whose numbers correspond or can be construed in whole number values. Well, both are usually considered protein standards, depending on your purpose.
Unstained protein ladders
Unstained protein ladders, just like the name suggests, are not usually visible during electrophoresis. To be seen, scientists must always stain them with the rest of the gel.
Prestained protein ladders
Unlike unstained protein ladders, prestained ladders are usually visible during the process of electrophoresis. Thanks to this phenomenon, the scientist or researcher can easily monitor the whole process of separating the protein sizes targeted for the session. Fortunately, these markers and ladders will remain visible even after the gel staining or membrane transfer for western blotting detection. Blue prestained markers are usually bonded to Coomassie blue dye, albeit non-covalently. This is the same dye used for gel staining.
Keep in mind that prestained markers are not as precise as the unstained proteins. This is because the molecular weight of prestained proteins is relatively more prominent than that of unstained protein. I hope that this makes sense. If you are going for commercial prestained protein markers, expect them to be multicolored. Other bands are also green, orange, and red apart from the usual blue color. This range of colors ensures that you get precise reference points, which will help you identify the band that gives the specific molecular weight.
This is an accurate method since you still get a consistent answer on whether some bands didn’t manage to stay at the bottom of the gel.
Western blotting (chemiluminescent)
We did mention western blotting at some point during our discussion. Well, consider that there are specific protein ladders for this phenomenon. Let’s take a look at these:
iBright Prestained Protein Ladder
Do you remember our discussion about prestained protein ladders? Well, this type is designed to offer direct visualization during electrophoresis and, as a result, the transfer of western blots. It also plays a massive role in the confirmation of western blot detection. This ladder has twelve recombinant proteins with different molecular weights.
Out of these, ten are blue-stained and have a fluorophore label, which allows direct and more advanced visualization. The remaining two are unstained to enable chemiluminescent and fluorescent detection through their IgG binding sites. To add to the last part, the detection captures both the primary and secondary antibodies required by the target protein.
Super Signal MW protein ladders
By now, you know that there are several types of protein ladders. Well, under this category, we have Thermo Specific SuperSignal Molecular Weight Protein ladder and the Super Signal Enhanced Molecular Weight protein ladder. Both are made up of eight active mixtures of recombinant proteins, all with different sizes.
In case you are wondering what is special about this class of protein ladders, here is the reason. Each of the eight proteins has a functional polypeptide sequence used to bind antibodies responsible for the western blot. If you choose to visualize these markers, you can use the recommended substrates when dealing with enzyme-labeled antibodies or fluorescent dye-labeled antibodies. This also comes with an individual pink tracking die, which will monitor the whole process and membrane transfer, saving you the need to have additional colored markers. Isn’t this what you want?
Lastly, these markers can be used with nitrocellulose, PVDF membranes, multiple blocking buffers, among many other detection methods.
Near infrared fluorescence imagers
Lastly, we will discuss protein ladders for near-infrared fluorescence imagers. These are generally made of ten proteins that are blue-stained. Some of them are also fluoro-labeled to serve their ole, which is fluorescent visualization, and protein sizing as electrophoresis occurs. The markers found in this specific category have sharp bands when a different analysis is done. These include Typhoon Imagers and LI-COR Odyssey Infrared Imaging System.
There are a lot of things that you need to know regarding electrophoresis. Protein ladders play an essential role in ensuring the success and viability of the process. Feel free to go through the article once more if you cannot easily digest our discussion.
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