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SDS-PAGE: The Easy Way to Find the Wells

If you have ever attempted to load a SDS-PAGE gel only to miss the well, stab the divider, and then watch helplessly as your sample squirts off into the wrong well, then this tip is for you.

The fortunate among us are able to use pre-cast gels with the wells outlined on the gel plate, but home-made gels don’t have this feature. I’ve seen labs with various loading guides printed off on acetate that they stick to the front plate of the gel before loading, which is better than nothing, but only shows you where the well is supposed to be, not where it is. It is true that after loading enough gels you start to develop an eye for finding the wells, but there is an easier way.

The trick is as simple as this: add a bit of bromophenol blue to your stacking gel. If you remember, bromophenol blue is already in your loading dye, so you aren’t adding anything new to the SDS-PAGE equation. I find that adding the dye to 0.003% is enough to color the stacker, but you can adjust the concentration to your liking. To make things even easier, I simply add the dye to my 4X Stacking Gel Buffer (0.5M Tris-HCl pH 6.8, in my case) to a final concentration of 0.012%. Now you don’t even have to add a new line to your recipe. You can see the results below.

 

Before loading the samples

Before loading the samples (Yes, I know these are ugly wells.)

Samples Loaded

Samples Loaded…

20 minutes into the run

…a third of the way into the run…

After an hour, close to the end of the run

…and close to the end of the run.

As you can see, the wells are easy to visualize. Once the voltage is applied, all the dye in the gel collapses down into the dye front and the gel runs normally. If the color of the stacking gel is too similar to the color of your protein samples, then you can simply add more bromophenol blue to your concentrated loading buffer (you’ll find that recipes vary on this point anyway). For the sake of the photographs, I ran this gel without the cover – this is potentially dangerous, so don’t try this at home…urr… I mean, your lab.

16 Comments

  1. Meny on March 19, 2019 at 1:11 am

    Nice trick. I am using it for many years. Even published it on YouTube 3 years ago.
    https://www.youtube.com/watch?v=hwZbX1H30i0

  2. biotef on February 28, 2018 at 6:01 am

    indeed a nice article. unfortunately i am unable to see the images. can anyone suggest the reason for it.

  3. Chen on April 11, 2010 at 10:35 am

    Hey Pavan,
    I’m using also a premade stacking/resolving mix (w.o the APS/TEMED) and in the past few months we encounter a problem that the stacking gel won’t polymerize/polymerize slowly. Moreover, the wells have shrank to their base, leaving me with useless gel!
    Have you encountered such a thing?
    10x,
    Chen

  4. SexComb on April 3, 2010 at 6:57 am

    In our lab we simply take a marker pen, and draw the position of the wells onto the glass when the comb is still in the gel. This way the wells are visualised precisely where they are. But this trick with the bromphenol blue is also neat.

  5. Tenzin Gocha on April 2, 2010 at 1:37 am

    Thank you Jode..I will try it out and see.

  6. Jode on April 1, 2010 at 3:22 pm

    Tenzin,

    You can use a modified version of John’s idea. Take some 1X sample buffer (or just some ~30% glycerol with bromophenol blue in it) and load every other well, and watch the empty wells. Then flush the sample buffer out of the wells carefully (ie- don’t detach the “fingers” while flushing).

    While you are at it, keep your camera handy and take a picture when you get a leaky gel. Send it to the company that made them and get some free gels out of them for your trouble.

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