SDS-PAGE: The Easy Way to Find the Wells |
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Jode Plank |
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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 (Yes, I know these are ugly wells.)
Samples Loaded...
...a third of the way into 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.
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Lis
Sweeeet idea.
Thanks Jode!
ps. I appreciate your humor (:
Peter
Wow! I’ll definitely try it out! We routinely use a marker pen to mark the borders of wells on the outer glass surface before pulling the comb out of the gel. It does not hurt either, however it takes a few more minutes hands on than just simply pouring the gel.
Mike Jones
I tried this tonight in lab, I like it. I never really thought finding wells was a big problem, but now that they are blue it’s just plain luxurious. That being said, I have some comments. I found 0.003% to be a little too much for my taste. I scaled it back to ~0.001%.
A word of note for others: It’s gonna look ALOT more blue in the bottle than in your v. thin gel. I was suprised how much more pale it looked when it was only 0.75mm thick
Yannick v.G.
Fantastic idea!
I’m actually running a gel this way right now. But as Mike pointed out, the solution is a lot darker in the bottle than in my gel (1,5mm). I added the bromophenol blue by feeling though, so i’ll add a little bit more.
Let’s take the occasion to discuss western blot “stock solutions”. I prepared a 1x bottle of Tris, water and SDS, so i just have to add Acrylamide and APS. Does someone have experience with stock solutions including APS or maybe acrylamide?
Jode
Yannick v.G.,
I’m glad you like the tip. APS is a strong oxidizing reagent that doesn’t play well with others, so I would leave it out of your master mix. Its lifetime at room temperature (or at 4°C, for that matter) is relatively short, so I would suspect that after a couple of days to a couple of weeks, your gels wouldn’t polymerize correctly, anyway.
You might have a better chance of including the acrylamide, if you stored the solution at 4°C. You might have to warm the master mix in order to get it to polymerize correctly, though. Also, I know that having SDS in a premade gel causes the gel to ‘expire’ more quickly – I don’t know if a similar reaction would take place in your solution. There is also a pH dependent degradation of acrylamide, but I don’t know if this would occur at 6.8 or not.
It seems like there are a lot of ways that including these two components could go wrong…
Sarah
Pyrogen G in the stacking gel works nicely as well – and you get a pretty blue and pink effect.
Sarah
Argh! By Pyrogen, I mean pyronin-G, apologies.
Pavan
Cool idea Jode! I love Bitesize Bio for all the creative suggestions
@Yannick v.G- I frequently make stock solution for stacking and resolving gel having SDS, Acrylamide and buffer and store at 4 degrees. APS and TEMED is added later. I have not seen any problems so far by using up to 1 month old stock solutions. I hope it help.
John
Weird. I was thinking about this earlier this week.
I just tried an alternative way of doing this: Remove the comb and fill the wells with diluted loading buffer (until it’s transparent blue), sit for a minute or so, then wash the wells out with water.
The residual loading buffer outlines the wells pretty clearly.
Just an idea for those that don’t want to add it to their stacking gel.
Thanks for the article Jode!
Tenzin Gocha
I have been using some precast commercial gels lately and i get a lot of leaky wells. Is there a technique or trick in checking if the wells are leaking .
Thank you in advance
Jode
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.
Tenzin Gocha
Thank you Jode..I will try it out and see.
SexComb
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.
Chen
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