Here is a time lapse of an SDS-PAGE gel, and a brief explanation of what it is.

This is a time lapse of an SDS-PAGE electrophoresis I did a long time ago

SDS PAGE is an acronym for a type of technique used to both purify and analyze protein samples by separating them by weight.

Here is a little crash course on proteins

Proteins are the main workhorse of the cell. They do everything from providing scaffolding for the cell, allow them to stick to each other, fight disease, and allow the chemistry of life to happen

To study these proteins, we need a way to separate them from the sample we have. This is where electrophoresis comes in to play.

In the image above, we see the structural levels of a protein. The only take away from this is that when amino acids stick together in to a string, called a polypeptide chain, the complexity of the folding that occurs can be divided down to different structure levels.

what does this have to do with electrophoresis? simple. If we are going to separate a sample of proteins by weight, which is often proportional to the length of the polypeptide chain, we have to level the playing field. This is done with a compound called SDS

SDS stands for sodium dodecyl sulfate, which is just a fancy detergent thats used to unfold, denature, proteins back in to their primary " string" form. If we dont use this, then purification wont be just by weight, but also by how hydrodynamic the protein is. Every proteins has a unique shape, some of which are bulkier than others. To remove the variable of shape, we add SDS.

Another thing that SDS does is that it gives the polypeptides a uniform negative charge that is proportional to its weight. This is what allows us to separate a sample by weight. The reason this is critical is because, just as with weight, different proteins can have different overall electrical charges. SDS negates that, and give its own, overall negative, charge.

So, how does separation happen?

To separate/purify our sample we first have to make our Polyacrylamide gel. Creating the gel isnt that hard, its the timing that critical. The reason why is the gel comes in powder form, and once everything is mixed, it has to be poured in to a mold before it becomes a gel.

At the molecular level, the gel acts as a sieve. As the polypeptide is pulled through, it interacts with the molecules of polyacrylamide. This is where the magic happens. if you have a large protein, it will have a large polypeptide chain, so it will interact with more fibers in the gel at a given time. As a result, it will move slower than a smaller peptide. As a result, this means the smaller, lighter peptides travel further.

but, how do the protein chains travel through the gel?

As you can see in the diagram above, the gel is placed in a special apparatus that applies a electric charge across the gel, with the bottom of the gel being the cathode, and the top being the anode. Remember how SDS give peptides an overall negative charge? well, when the voltage is applied, the peptides are yanked towards the cathode. This is what drives the process

we can alter concentration of the gel to get better, more defined separation, but that only increases your run time, and no one wants to stay in the lab more than they have to.

But, how do we know when separation is done?

Remember that blue band moving across the gel in the gif? this is a special dye that has a negative charge that moves with the protein sample. Its mixed in with the protein sample, and gives a visual indication of how far the sample has traveled.

ok, great. we've run the sample. How do we analyze it?

Most SDS-Page gels are run with a standardized sample called a protein ladder. This is just a sample containing proteins of known weights. Its put through the sample protocol as your sample of interest, so when its run, it will separate out in a similar manner. The purpose of this ladder is to allow us to quickly determine the possible weight of the protein we are analyzing, and to help locate were our protein of interest in the gel based on the weight.

once we get our gel, we will imerse it in a dye that binds to all proteins in the gel, and we get a banding pattern as seen in the 3rd picture.

I have a dried gel from a run I did in my research notes, but I didnt have time to find it. If you guys want, I could try to find it an post it.

The ladder only provides crude analysis by weight. If we really want to analyze our sample run, and locate our protein of interest, we can go a step further.

What we are looking at above is called a Western Blot. This is the step above SDS-PAGE. The way this works is we do a sort of secondary electophoresis where the separated proteins are transferred to a special paper (I dont remember the name, but all i know its pretty combustible)

From there, we treat it with antibodies that specifically target the protein of interest. once that is done, we have to apply a secondary agent to help "illuminate" the bound protein.

In the olden days, they used to use radiolabelled samples that emitted radiation. This radiation was then captured using special film. The trick with this was getting the exposure right so we could easily see the banding. The lab I used to work at used that technique, so I got to do it. I actually have an exposure that I kept in my notes. I didnt have time to find my old research notes, but if anyone wants to see the picture, let me know.

The more modern method that I used at the time, and at my persistence to help get the lab to this century, was to use either antibodies labelled with a fluorescence tag or horseradish peroxidase. In essence, samples treated with either antibody will emit light, which is then captured by a scanner. The advantage of this is we can not only see the bands more clearly, we can do analysis of each band to see how much sample was there.

In the picture above, we were analyzing the level of a particular enzyme that was the target of drug trial. The dark band is the intact enzyme, while the subsequent bands are just fragments of the enzyme that broke down. We know all of the bands in this picture are of just that enzyme because of how we use an antibody that first only bound to the enzyme before adding our secondary antibody.

Western blots are used in medicine to help diagnose conditions such as AIDS, which is looking for the HIV proteins, or in diagnosing blood disorders, such as thalasemia.

well, I hope this was educational. I was meaning to do a more detailed version of this but I have some prior engagements to attend, so here is a crash course. Post whatever questions you might have.

Here is a link to the timelapse I made. At the time, no one had posted time lapses of a running PAGE: https://youtu.be/bwQ7aTW7FpI