How SDS-PAGE works

by on 18th of September, 2008 in Protein Analysis, Detection & Assay
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About Nick Oswald
Nick Oswald started Bitesize Bio on a Macbook on his kitchen table in 2007 while in his 7th year of working as a molecular biologist in biotech. He made it his day job in 2010 and has been loving it ever since.

SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) is commonly used in the lab for the separation of proteins based on their molecular weight. It's one of those techniques that is commonly used but not frequently fully understood. So let's try and fix that.

SDS-PAGE separates proteins according to their molecular weight, based on their differential rates of migration through a sieving matrix (a gel) under the influence of an applied electrical field.

Making the rate of protein migration proportional to molecular weight.

The movement of any charged species through an electric field is determined by it's net charge, it's molecular radius and the magnitude of the applied field. But the problem with natively folded proteins is that neither their net charge nor their molecular radius is molecular weight dependent. Instead, their net charge is determined by amino acid composition i.e. the sum of the positive and negative amino acids in the protein and molecular radius by the protein's tertiary structure.

So in their native state, different proteins with the same molecular weight would migrate at different speeds in an electrical field depending on their charge and 3D shape.

To separate proteins in an electrical field based on their molecular weight, we need to destroy the tertiary structure by reducing the protein to a linear molecule, and somehow mask the intrinsic net charge of the protein. That's where SDS comes in.

The Role of SDS (et al)

SDS is a detergent that is present in the SDS-PAGE sample buffer where, along with a bit of boiling, and a reducing agent (normally DTT or B-ME to break down protein-protein disulphide bonds), it disrupts the tertiary structure of proteins. This brings the folded proteins down to linear molecules.

SDS also coats the protein with a uniform negative charge, which masks the intrinsic charges on the R-groups. SDS binds fairly uniformly to the linear proteins (around 1.4g SDS/ 1g protein), meaning that the charge of the protein is now approximately proportional to it's molecular weight.

SDS is also present in the gel to make sure that once the proteins are linearised and their charges masked, they stay that way throughout the run.

The dominant factor in determining an SDS-coated protein is it's molecular radius. SDS-coated proteins have been shown to be linear molecules, 18 Angstroms wide and with length proportional to their molecular weight, so the molecular radius (and hence their mobility in the gel) is determined by the molecular weight of the protein. Since the SDS-coated proteins have the same charge to mass ratio there will be no differential migration based on charge.

The gel matrix

In an applied electrical field, the SDS-treated proteins will now move toward the cathode at different rates depending on their molecular weight. These different mobilities will be exaggerated the high-friction environment of an gel matrix.

As the name suggests, the gel matrix used for SDS-PAGE is polyacrylamide, which is a good choice because is chemically inert and, crucially, can easily be made up at a variety concentrations to produce different pore sizes giving a variety of separating conditions that can be changed depending on your needs. You may remember that I previously wrote an article about the mechanism of acrylamide polymerisation previously – click here to read it.

The discontinuous buffer system and the stacking gel – lining them up at the starting line.

To conduct the current through from the anode to the cathode through the gel, a buffer is obviously needed. Mostly we use the discontinuous Laemmli buffer system. "Discontinuous" simply means that the buffer in the gel and the tank are different.

Typically, the system is set up with a stacking gel at pH 6.8, buffered by Tris-HCl, a running gel buffered to pH 8.8 by Tris-HCl and an electrode buffer at pH 8.3. The stacking gel has a low concentration of acrylamide and the running gel a higher concentration capable of retarding the movement of the proteins.

So what's with all of those different pH's? Well, glycine can exist in three different charge states, positive, neutral or negative depending on the pH. This is shown in the diagram below. Control of the charge state of the glycine by the different buffers is the key to the whole stacking gel thing.

So here's how the stacking gel works. When the power is turned on, the negatively-charged glycine ions in the pH 8.3 electrode buffer are forced to enter the stacking gel, where the pH is 6.8. In this environment glycine switches predominantly to the zwitterionic (neutrally charged) state. This loss of charge causes them to move very slowly in the electric field.

The Cl- ions (from Tris-HCl) on the other hand, move much more quickly in the electric field and they form an ion front that migrates ahead of the glycine. The separation of Cl- from the Tris counter-ion (which is now moving towards the cathode) creates a narrow zone with a steep voltage gradient that pulls the glycine along behind it, resulting in two narrowly separated fronts of migrating ions; the highly mobile Cl- front, followed by the slower, mostly neutral glycine front.

All of the proteins in the gel sample have an electrophoretic mobility that is intermediate between the extreme of the mobility of the glycine and Cl- so when the two fronts sweep through the sample well the proteins are concentrated into the narrow zone between the Cl- and glycine fronts.

And they're off!

This procession carries on until it hits the running gel, where the pH switches to 8.8. At this pH the glycine molecules are mostly negatively charged and can migrate much faster than the proteins. So the glycine front accelerates past the proteins, leaving them in the dust.

The result is that the proteins are dumped in a very narrow band at the interface of the stacking and running gels and since the running gel has an increased acrylamide concentration, which slows the the movement of the proteins according to their size, the separation begins.

What was all of that about?

If you are still wondering why the stacking gel is needed, think of what would happen if you didn't use one.

Gel wells are around 1cm deep and you generally need to substantially fill them to get enough protein onto the gel. So in the absence of a stacking gel, your sample would sit on top of the running gel, as a band of up to 1cm deep.

Rather than being lined up together and hitting the running gel together, this would mean that the proteins in your sample would all enter the running gel at different times, resulting in very smeared bands.

So the stacking gel ensures that all of the proteins arrive at the running gel at the same time so proteins of the same molecular weight will migrate as tight bands.

Separation

Once the proteins are in the running gel, they are separated because higher molecular weight proteins move more slowly through the porous acrylamide gel than lower molecular weight proteins. The size of the pores in the gel can be altered depending on the size of the proteins you want to separate by changing the acrylamide concentration. Typical values are shown below.

For a broader separation range, or for proteins that are hard to separate, a gradient gel, which has layers of increasing acrylamide concentration, can be used.

I think that's about it for Laemmli SDS-PAGE. If you have any questions, corrections or anything further to add, please do get involved in the comments section!

21 thoughts on “How SDS-PAGE works”

  1. Steve says:

    Thanks, this is very informative and now I don't feel like such a moron for doing something that I didn't really understand.

  2. Anne says:

    I finally understood the principle behind the stacking gel pH. Thank you!

  3. DK says:

    I feel that the stacking effect is not adequately described (also see "*" below). At least in my extensive experience with most grad students and postdocs who usually barely remember any physics. Here, let me try:

    1. Yes, fast Cl- ions run away, slow glycine- ions that are even further slowed by the fact that large proportion is them is not even ions half the time (pH is chosen so!), come into the stacking gel.
    2. Since electric current = rate of charge movement ==> fast ions in the gel = high conductivity/low resistivity and slow ions in the gel = low conductivity/high resistivity.
    3. The rate of electrophoretic movement is a linear function of E, electric field intensity (mobility = some coefficient x E). E is a "voltage gradient", i.e. E=V/distance.
    4. Recall Ohm's law, V = I x R. In our case, we have a linear circuit so that R is a sum of resistivity of resolving gel (Rr, low) and resistivity of stacking gel (Rs, high): V = I x (Rr Rs). So the overall voltage that comes from the power supply can be said to consist of a sum of two voltages: V= I x Rr I x Rs = Vr Vs (or, in physics parlance, overall voltage "drops" unequally on resolving and stacking gels). The voltage drop on a stacking gel is higher because it has higher resistance (e.g., few charges that move slowly).
    5. So, for as long as we have enough buffering in the stacking gel, the situation is this: higher voltage drop on a stacking gel and the voltage drops on a shorter distance. Which means that the driving force of electrophoresis, E (which is, again, voltage drop over distance) is much higher in the stacking gel.
    6. The stacking gel is made of very low percentage of acrylamide so that proteins move in it roughly irrespective of their molecular mass. Since in the presence of SDS their charge to mass ration is approximately equal, they all move in the stacking gel about the same – and FAST, comparing to how any of them moves in the the resolving gel.
    7. Once the particular protein molecule reaches the resolving gel, its rate of migration slows down dramatically (lower E in the resolving gel and higher percentage of AA in the resolvign gel). So what we get is a situation where all proteins loaded on a gel initially move fast untill they reach resolving gel where they slow down – and this allows protein molecules that were initially behind them (and thus still moving fast) to catch up. In other words, the initial wide band of loaded sample gets concentrated in the narrow band on the boundary between stacking and resolving gels.
    8. From there it's simple: in the higher % AA resolving gel proteins of different mass move with different rate because of the friction against the with a gel, thus allowing the resolution approximately according to their molecular mass.

    Few practical conclusions that follow from the above:
    - That is why you should never adjust pH in the running buffer. Doing that adds fast ions which would move into the stacker and make stacking less efficient.
    - That is why it is not recommended to load samples with high salt content. High salt usually = more fast ions ==> again, screwing up the way stacker works.
    - That is why gel loading buffer has the same buffer composition as the stacker (pH 6.8 – which isn't, BTW, great for heating proteins because some proportion of them undergoes acid hydrolysis).

    * This statetement in your original description is simply incorrect:
    So the glycine front accelerates past the proteins, leaving them in the dust.

    This makes an impression that proteins somehow are left without glycine at all. That's not true – glycine molecules are continuously coming from the running buffer. What changes with glycines hitting separating gel is that they start running faster, ensuring that the conductivity of the resolving gel remains high – something that, as explained above, is essential for the stacker to work its best.

  4. Avatar of Nick Oswald Nick Oswald says:

    DK – thanks for providing such an informative comment. Hopefully your points will clear up any questions people are left with at the end of my article.

    I hadn't intended to give the impression that the proteins are left without glycine ions in the resolving gel, but that the glycine front speeds up in relation to the protein front after leaving the stacking gel. But, reading it again, I can see that it could be read that way. Thanks for making a good point – again, I hope your comment clears things up for anyone who got the wrong idea.

  5. Monisha says:

    Wot an awesome website!..a lotta fundamentals which the teachers dun ever discuss(i'm unsure if they know) got cleared :)

  6. Avatar of Nick Oswald Nick Oswald says:

    Monisha — tell your friends! :)

  7. Lekhana says:

    Hi,

    I'm deviating a little from the current topic here. Can you tell me what is the function of ethylene glycol in a Conformation sensitive gel electrophoresis (CSGE)for detection of mutations in DNA. I know it used as a denaturant but what is the exact interactions with DNA that actually help in denaturing it?

  8. jithendran says:

    Awesome explanation simple and usefull…….

  9. Axel says:

    Hey,

    Nice explanation of the SDS-PAGE, I just have to link your post to my work fellows when they have this question.

    I might be wrong, but in this setup the anode goes for the positive electrode (and cathode for the negative one), as far as the SDS-bound molecules are negatively charged and are called anions and as far as anions go to the anode (the red electrode on the generator outlet).

  10. Seena says:

    That was a very interesting description of SDS Page.It sure has improved my understanding of SDS Page.
    Thak a lot Nick

  11. Brooke says:

    Wow, just wow. This man deserves a medal! Call me crazy, but I've cited a couple of your basics pages in second year university assignments. It's so hard to find good basic information and when you do you find that 50 year old articles are going for $30!!! I don't care if they dock marks, I think you're a wonderful source of decent, comprehensive info to people just starting out and I hope more people make their way here.

    Thank you so much!!!

  12. Avatar of Rashid Rashid says:

    Thanks for such article. It increases the knowledge and gives lot of clarification. Now onwards I will be the frequent user of this website.

  13. Hi,

    I've been having some problems with SDS-PAGE lately in that I seem to be getting accumulation of protein at the interface of stacking gel and running gel even after the gel has run its course. Would you know why this is?

    Also I seem to get a similar flat line above my bottom marker band, so smaller proteins are accumulated in the flat line rather than nicely separated bands.
    Thanks

  14. Avatar of labman labman says:

    Did anyone of you try out the method described in this paper?

    "Polyacrylamide Gel Electrophoresis without a Stacking Gel: Use of Amino Acids as Electrolytes" Ahn et al. 2001

  15. Avatar of NSM NSM says:

    Very nice explanation, thank you!

    I'd just like to say that the link for the tutorial is broken.

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  17. Avatar of henwangen henwangen says:

    Can you please help me with the following question:

    "You have purified an enzyme that is composed of 3 subunits. Two of these subunits have a molecular weight of 28kDa and the other is of 14 kDa. This enzyme is run on an SDS-PAGE. You made a mistake and instead of making a 12% acrylamide gel, you made an 8% gel. Where wll the enzyme subunits migrate in this case? Explain why?"

    I know that the higher molecular weights the lower the % gel. Will the 28kDa subunit migrate as normal, while the 14 kDa subunit will migrate slower?

  18. awesome explanation…….just went right into my brain without any doubt

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