Once the proteins have been denaturated, they can be separated according to size by SDS-PAGE (ahem… “sodium dodecyl (lauryl) sulphate-polyacrylamide gel electrophoresis”. Simple as that.). SDS is a strongly anionic detergent. It binds quantitatively to the proteins and overpowers any charge the proteins may possess, forcing identical negative charge onto all proteins. This eliminates the risk of the individual charge of the proteins interfering with the separation. In addition, SDS also strengthens the earlier denaturation step.

Collectively, these approaches enable separation of the proteins based solely on their sizes. When the gel is placed in a tank filled with a buffer and an electric current is applied, the negatively charged proteins migrate through the gel, towards the positively charged end. As was explained above, smaller proteins encounter less resistance, and therefore wander more rapidly than larger proteins. When the electroblotting is finished, the proteins are separated.

Graphically, this migration results is a number of different bands on the gel, each band corresponding to a different molecular weight. Usually, a ladder is also loaded onto the gel next to the protein samples. The ladder is a mix of proteins with known molecular weights, and can therefore be used as a reference, to which you can compare your own samples.

The bands are usually measured in kiloDalton, kDa.

 

A western blot with myostatin to the left and a ladder (CruzMarkerTM) to the right. With the ladder as a reference, the strongest myostatin band can be determined to have a molecular weight of approximately 42kDa.

 
In a way, these separated protein bands are the final result, at least if you only are interested in determining whether or not the protein is actually present in your sample. 

However, there is a small catch. They are not yet visible! Hence, the next step is to make them that…

 
 
But hey… wait a minute. What do you mean with a gel? What is that?  
 

Well, a gel is exactly what it sounds like. Something you use to mess up your hair with, but held into place by two glass plates. Oh, and the contents may be slightly different…  

You can make the gel by yourself, or buy it precast and ready to use. Like everything else in the world, it is a matter of time, money and personal preference. In a Western blot, the most frequently used type of gel consists of a polyacrylamide matrix. Polyacrylamide is a cross-linked polymer of acrylamide, and there are gels available in several different concentrations of acrylamide, sometimes even with a gradient (which means that for instance, the top of the gel can have a concentration of 3%, which increases to 15% in the bottom of the gel).  

 
 

Okay, fine, but if you can’t see the proteins, how do you know when the separation is finished? What if they wander off the gel? 

 

It is important to keep time, so that the proteins do not migrate too far. To do this, you need to add an agent that stains the samples. Bromophenol blue is popular to use for this purpose. When mixed with the sample, it wanders in front of the proteins on the gel. In addition to keeping track of the migration, it also simplifies the task of checking whether you really applied your sample into the well and not beside it.  

Laemmli sample buffer® is a commercially available product especially designed for protein sample preparation. In addition to bromophenol blue, it also contains 2-mercaptoethanol (for 3D-structure breakage), SDS (to keep the proteins straightened out and give them a uniform charge) and glycerol (to increase the density of the sample, thereby making it easier to apply the sample into the well). 

Another option to staining (or even better, to be used parallel to it) is to run a colour ladder in one of the empty wells. It works in the same way as the protein ladder mentioned earlier in this chapter, although the proteins bands are coloured in different colours which are all visible directly on the gel. 

 
 

How do I know what the protein concentration should be in my samples in order to get as good results as possible?

 

Which concentration that is optimal (i.e. gives the best balance between band signal and background) for the protein of interest is often hard to calculate beforehand. A good idea is to devote one of the first Western blot runs to determining it! Simply make a series of dissolution and apply different concentrations into the wells. When your blot has been developed (the last step of the protocol), you should get a good indication of which sample concentration that will work best for your protein.

 
A series of dissolution has been applied in order to find the optimal protein concentration. 1:3 dilution (~5.3 µl) will be chosen from this series.