Why Is SDS-PAGE Run Vertically? Here are 3 Great Answers

SDS-PAGE gels are a bit tricky to put together.

Make the resolving gel, pour, add isopropanol, wait, wipe isopropanol away, make the stacking gel, pour, wait—Blimey!

Besides, if you don’t get the seal just right, the unpolymerized gel leaks, and then you have to start all over again.

Hang on—why is SDS-PAGE run vertically anyway?

It’d be way simpler to have a horizontal SDS-PAGE system. After all, agarose gel electrophoresis runs horizontally and never leak or tear.

And how different are they really? Why is SDS-PAGE run vertically?

We’ll answer those burning questions in this nifty article and then, for fun, play devil’s advocate and consider some suggestions left in the comments section.

Why Is SDS-PAGE Run Vertically?

Of course, life would be much simpler with a horizontal SDS-PAGE system, but there are 3 excellent reasons why it has to be vertical.

Answer 1: SDS-PAGE Gels are Discontinuous

As you well know from hours spent pouring them, SDS-PAGE gels are two-component gels.

They comprise a stacking gel and a resolving gel.

A vertical arrangement allows you to make them sequentially. You pour the resolving gel first, and then once it is set, pour the stacking gel on top of it.

This results in one contiguous gel.

It would be difficult, if not impossible, to prepare such a gel horizontally.

Answer 2: Gel Polymerization Is Inhibited by Oxygen

Stop!

Before you run down the patent office clutching technical drawings of brilliant new casting apparatus on the back of a beer mat, consider this.

Oxygen inhibits the polymerization of SDS-PAGE gels.

Specifically, ammonium persulfate (APS) in the SDS-PAGE gel mixture reacts with TEMED to produce sulfate free radicals.

These free radicals react with the acrylamide molecules in the presence of TEMED to initiate polymerization.

Molecular oxygen in the atmosphere reacts with sulfate free radicals, however. And if there are no sulfate free radicals available to initiate acrylamide polymerization, there’s no gel—just the unset mixture of ingredients.

Sandwiching the gel mixture between two glass sandwich plates and isopropanol/a comb protects it from those pesky oxygen molecules.

A horizontal system would inevitably be:

1. Exposed to the atmosphere and the polymerization reaction wouldn’t proceed efficiently.

Or:

2. Extremely difficult to pour with a straight interface between the resolving and stacking gels.

It looks like Bio-Rad will keep the SDS-PAGE gel casting market cornered for now.

Answer 3: A Vertical System Is Cheaper

Money always finds a way to have some influence.

The reagents used in SDS-PAGE gels are relatively expensive.

So, it wouldn’t be cost-effective to pour them like agarose gels because you’d need much thicker gels to accommodate your sample analytes.

It’s more cost-effective to make thinner gels with tall but thin sample wells. And this is redoubled if you take a moment to consider how many SDS-PAGE gels a busy laboratory can get through.

What’s the Difference Between Agarose and Polyacrylamide Gels?

I won’t waste your time and labor any points here. Here are their key differences:

1. Polyacrylamide gels polymerize. Agarose gels set;
2. Agarose gels contain a fluorescent dye. Polyacrylamide gels do not;
3. Polyacrylamide gels are discontinuous. Agarose gels are continuous;
4. Agarose gels may be re-run. Polyacrylamide gels may not;
5. Polyacrylamide gels separate a narrower range of analytes;
6. Agarose gels don’t contain any toxic ingredients!

For Argument’s Sake

The question “why is SDS-PAGE run vertically?” is not uncommon because a few fun suggestions have been left in the comments section since this article was published. And here at Bitesize Bio, we love to engage with our readers!

Let’s consider these suggestions and whether or not they could work in practice.

“What about pre-cast gels?“

Unfortunately, you can’t buy horizontal pre-cast SDS-PAGE gels. And even if you could, there’s no gel tank on the market in which you could run it!

“…Could You Not Pour [a Gel and Move It] Horizontally after It’s Set?”

Definitely!

You could load an SDS-PAGE gel that you’ve cast following a standard protocol and then flip it horizontally. And there’s nothing to stop you from placing the gel, sandwiched between the glass plates, into an agarose gel electrophoresis tank and running it horizontally.

But.

You’ve not really saved yourself any hassle by doing this, so what’s the point? You’ve still cast and loaded the gel vertically.

And I’ve never run an SDS-PAGE gel in an agarose gel tank. Nor do I know anybody who has tried. So I can’t say whether it would work or not.

Plus, whilst I bet you could load the sample onto a horizontal SDS-PAGE gel using those bendy gel loading tips, what’s to stop your sample from spilling out?

It might be the case that 0.75-mm thick gels can retain your sample by surface tension alone when horizontal, but thicker gels would almost certainly leak!

Don’t forget too that the anode running buffer needs to have a single point of contact with the cathode running buffer!

This is why we use a buffer dam when running one gel and is what causes the analytes to migrate downwards.

For similar reasons, horizontal gels almost certainly wouldn’t work for tris-tricine SDS-PAGE because the anode and cathode buffers must be kept separate and have a different composition.

“…Having [the Gel] Vertical [Requires] More Buffer…”

It certainly does.

But, as the commenter correctly pointed out, the SDS-PAGE running buffer is cheap. It may also be re-used several times.

Given the compelling answers that we’ve just presented to the question “why is SDS-PAGE run vertically?” saving money on running buffer is not enough reason to change the incumbent vertical system.

A Note from One of Our Readers

Since this article was republished, a reader contacted the Bitesize Bio team to clue us in on a legacy SDS-PAGE system that did run horizontal gels. [1]

It was called “PhastSystem.” You can read the user manual here. There’s even a picture of a stained gel that was run horizontally! Apparently, the method used super-thin gels, which sped up running, blotting, and probing.

It doesn’t look like it’s available anymore (although one of our team did find one for sale on a well-known auction site).

Bad news for anyone who wanted to try running horizontal SDS-PAGE gels. Good news for us because we don’t have to take the article down.

Thank you, reader!

And Now You Know

The next time somebody asks you “why is SDS-PAGE run vertically?” you can give a cogent answer!

Thank you for the comments. They have been fun to consider! Please feel free to chime in with your own suggestions, serious or not. It’s good fun. Be sure to let us know if you’ve found this article helpful, too.

And finally, be sure to check out some other great resources on SDS-PAGE here on Bitesize Bio. You can learn the differences between setting constant current or voltage. Or get right back to basics and learn how SDS-PAGE works!

Are you sick of leaky gels and wonky wells? Download our free SDS-PAGE gel recipe and casting protocol cheat sheet. It works—at any percent.

Originally published July 2013. Reviewed and updated February 2022.

Reference

Olsson I, Axiö-Fredriksson UB, Degerman M, and Olsson B (1988) Fast horizontal electrophoresis. I. Isoelectric focusing and polyacrylamide gel electrophoresis using PhastSystem. Electrophoresis 9:16–22