There are two categories of commercially available protein ladders, unstained protein ladders and prestained protein ladders. However, if you're wanting to know what the difference is between the two, you've come to the right place.
The difference between an unstained protein ladder and a prestained protein ladder is that unstained ladders require additional staining post electrophoresis. Prestained ladders are prestained with dyes for better visualization and tracking during electrophoresis.
A helpful way to understand the difference between unstained and prestained protein ladders is to use an analogy.
Imagine you have a few dogs, all are white. And you let your dogs out into a big field to run around. But it’s wintertime, and the field is snow covered.
As your dogs run off further and further, not only will it be hard to see them, but it’s going to be hard to see how far away each one is from you.
But what if you put different colored coats on each dog?
The individually colored coats help you track your dogs from a distance – the color of the cloth prevents you from losing sight of them even against the background of the snow.
Further, because each dog was assigned his or her own individually colored coat, you know exactly which dog is which.
The dogs represent the polypeptides, the stains are the differently colored garments.
Now, comparing this analogy with an unstained ladder, it would be as if you let your dogs out without any colorful coats – or dye.
So, you cannot discern them readily, until you catch up to them. This idea would be similar to staining the gel with Coomassie after you’ve done gel electrophoresis.
Table 1 below highlights the differences, advantages and disadvantages of prestained versus unstained protein ladders.
Table 1. Comparison of prestained versus unstained protein ladder.
|
Property |
Prestained Protein Ladder |
Unstained Protein Ladder |
|
Constituents |
Different sized polypeptides, each tagged with a colored dye. |
Different sized polypetides – Not tagged with dyes. |
|
Visualization in the gel |
Yes |
No |
|
Visualization in the membrane prior to Western Blot |
Yes |
No |
|
Can track how much electrophoresis took place |
Yes |
No |
|
Can track gel-to-membrane transfer for Western Blot |
Yes |
No |
|
Molecular weight determination? |
Yes. Less efficient. |
Yes. Very accurate |
In this article we will answer the following question – what is the difference between an unstained and a prestained protein ladder? In addition to this, we will take you through the advantages of prestained and unstained protein ladders, and how you can figure out which one is best for you.
In this article:
Advantages of using a prestained protein ladder:
Advantages of using an unstained protein ladder:
Prestained protein ladders
Protein ladders are composed of a series of bands, which are highly purified polypeptides of different well-defined sizes. These polypeptides get resolved during electrophoresis as separate bands - based on their molecular weights.
In a prestained protein ladder, each of these bands, or polypeptides, are already stained with a dye. What this means is that before even loading the ladders into the gel for electrophoresis, these ladders are previously stained and ready to go.
During electrophoresis, the polypeptides of the ladder show up in the gel as distinctly colored bands.
The cool thing about this is that even before the gel has completely run and then is further stained with a gel staining dye – Coomassie as in most cases – the ladder bands can be visualized by the experimenter.
Each band represents a molecular weight which is a combination of the molecular weights of the corresponding polypeptide and the associated dye.
Remember the white dog analogy where your white dogs are all wearing different colored coats – coats that represent a prestained ladder. Just as the dye adds weight, or bulk to the protein bands, distorting their exact molecular size, the bulk from the coats your dogs are wearing might also distort their shape and size.
In figure 1, each band of the ladder shows up in the gel, during electrophoresis, due to the colored dye tagged to it.
By comparing what you see in the gel with the reference information supplied by the manufacturer (like these GoldBio ladders), you can estimate the size of the polypeptides in your sample.
Figure 1. Prestained
protein ladders.
Advantages of using a prestained protein ladder:
Since the bands of the ladder show up while the gel is running, the main advantage of a prestained ladder is that they let you estimate how much your gel has already run – and depending on the necessity of the experiment – how much more time the electrophoresis needs to continue.
Here’s another way to think about it. Imagine you are experimenting with a new cake recipe. You’re not exactly sure how long it needs to cook, so you will need to continuously monitor its progress.
If your oven has a clear window and a light, the window and interior light lets you visualize the progress of your cake as it cooks. As you see the cake rise in the pan and brown just right, you know it might be time to take it out, or at least check the middle with a toothpick to see it is done.
Likewise, a prestained ladder allows continuous monitoring while your proteins are running.
Let’s break this down a little more.
The prestained ladder has polypeptides with a range of molecular weights. During electrophoresis, these polypeptides migrate to different distances
As the polypeptides migrate, researchers are able to watch real-time progression due to the dye.
Real-time monitoring allows researchers to see how much separation has taken place between polypeptides of different sizes.
Another advantage of prestained ladders is that they serve as a great read-out to monitor transfer efficiency when the electrophoresed polypeptides are transferred to a membrane for Western blot.
The brighter and clearer the ladder bands on the membrane following gel-to-membrane transfer procedure, the higher the efficiency of transfer.
Figure 2. Pictorial representation to show how a prestained ladder helps to track gel-to-membrane transfer during western blot
In this way, prestained ladders save experimenters the extra step of membrane-staining prior to Western blotting.
If the ladder was not prestained, the only way the experimenter would know whether good gel-to-membrane took place is by staining the membrane with a dye such as Ponceau.
Since a prestained ladder already shows up on the membrane due to the colored tags, the experimenter, just by seeing how bright or dim the ladder bands show on the membrane, would be able to gauge transfer efficiency even without Ponceau staining.
Unstained protein ladders
Unstained protein ladders are protein ladders in which the bands or polypeptides, are not prestained and therefore cannot be visualized while the gel runs. Instead, visualization takes place after the entire gel is stained with a dye such as Coomassie.
However, because no additional stain is added during electrophoresis, the representative weight of the polypeptide is more accurate. That is to say, the size each band of the ladder represents is solely the molecular weight of the corresponding polypeptide constituent.
Advantages of using an unstained protein ladder:
Unstained protein ladders have a huge advantage when it comes to accurately estimating the molecular weight of your protein bands since there are no dyes adding to the total weight of each ladder band.
So, the ladder bands are sharp – allowing very precise molecular weight determination of the proteins in the experimental sample.
