As part of my western blotting procedure, I ran my SDS-PAGE electrophoresis. Usually, I load in 2 lanes, 1 ladder (spectra broad range protein ladder) and my sample (sample lysate + Laemmli buffer + 2-mercaptoethanol). I load 5 ul and 20 ul, respectively. Then I run the electrophoresis at 200V, which has worked fine for me so far.
But recently, I followed the same procedure except that I loaded an extra ladder (biorad, SDS PAGE low MW marker, 5ul) and loaded 3 more lanes with samples (same contents, 20 ul each). So the only difference was that I loaded 6 lanes instead of the 2 last few times. I ran the SDS-PAGE at 200V but saw the bands tilt and become a "frowning" pattern, as seen in the attached photo. I saw similar questions being answered online and most said the solution was to lower the voltage. But 200 V had worked fine for me in the past; would adding more lanes (and thus samples) lead to this result?
Thank you!
Hi!
As long as I know, I think that maybe is a problem of your proteins because the ladder looks good.
I agree with Oscar, I feel there is a problem in sample preparation
Did you remove the sticker strip from the bottom of the gel? If the sticker is not removed, the outcome is usually poor.
Hi hye
i just have a couple of observations - in the second picture, i assume that’s when your samples start to run. I can see your samples and also the marker are like squeezing each other, and that can happen because A) the acrylamide % is too high, so your samples can’t travel properly in the pores of the gel, and B) the voltage is too high. And both options can make each other worse, if your samples can’t migrate properly trough the gel and you apply very high voltage, then your samples will be stuck and squeeze, which is similar to what i see on the pictures. Obviously at the end they will migrate, but not properly stacked in the pores of the gel. I can see you use precasted gels (if i am not mistaken), and therefore i assume changing the acrylamide % is not ab option for you. Although it will be worth to mention if you use gradient gels or single %. I would suggest change the voltage, there is also lots of bubbles there, adding more samples technically adds more sds from your buffer, although this shouldn’t be a big problem. I used 200v in my westerns, but only for very small proteins such as 22kDa And because i make my own gels. Unless you have changed something in sample preparation, your gels are pretty standarised so i would discard that possibility. You could also do a comassie staining of those gels, and see what shape the proteins have in the gel and discard possibilities. One thing you could also try is a lower voltage at the beggining of your run and then increase it when you are half way through. The reason is because your samples have to pass from stacking to resolving gel very very well, otherwise the full electrophoresis will be wRong. Give your samples some space at lower voltage to run properly, and then you can increase it. Hope this helps!
a) Air-bubbles are a big hindrance in SDS-PAGE and western blotting.
b) Run your protein gels at constant current (not to exceed 30-35 mA for mini gels and you may go upto 50 mA for large gels).
c) Make loading volumes same for all samples. This you can achieve by adding water/buffer to protein samples.
Hi that means gel didnt take enough time to solidefied and electrophoresis mast be forlonger time and lower voltage
Hi, in the first picture, the percentage of stacking gel needs to be optimized. In the second picture, the voltage also needs to be corrected and the tank must be kept in enough ice to remain cool during SDS-PAGE
Hello everyone!
Thank you all so much for your helpful comments!
I will take into account your inputs and incorporate them into my trouble-shooting for my next SDS-PAGE. I think my main problem lies in the gel percentage and the high voltage. I will try to opt for a lower acrylamide gel (in the 4-6% range) or a gradient gel as 10% may have been too high and not optimal.
Also, I'll try starting my electrophoresis at a lower voltage (such as 50-60V) for the first 30 minutes to help give the proteins in my sample ample time to line up in the stacking gel before increasing it to around 100 V once in the resolving gel.
And I'll also try keeping the container cool by adding an ice container inside.
That’s great hye. JUSt as a reference- i use 8% acrylamide for a protein of 150K mw, and 12% for my 22 kda protein. Hope next time goes better!
It looks as though you are using Pre-Made gels? Make sure that the SDS in the running buffer is good. If it is too old, the bands will run oddly. I have seen Gull Wing types of bands and various other shapes as well.
You should also add sample buffer only with NO protein in the empty wells. This will help to reduce the spread of the outer well protein samples.
Make sure that you have made all your Buffers from Fresh Stocks.
I agree with Philip Swigart , make sure your SDS running buffer is prepared properly, better to prepare it fresh when you run your gel nex time. I generally run SDS-PAGE between 95-130V. 200V may be too much which may be harmful to your gel.
I attached two figures one with bad SDS running buffer, the other with fresh SDS buffer. These images were obtained from the same lysates with the same running conditions except the SDS running buffer (wet transfer, probed with the same antibody, same amount of time).
Good luck.
- Make sure there are no air bubbles in the wells. I use a tuberculin syringe to rinse the wells with running buffer before loading the samples, this removes not only bubbles but also remnants of the polymerisation process.
- Samples should have low conductivity and buffering capacity, both should be the same for all samples.
- Use 1 x sample buffer to ensure that all wells contain the same volume of sample.
- Minigels are run with 10 mA constant current while the front is in the stacking gel, once it has entered the separating gel increase to 20 mA.
- Make sure to use the cooling loop of your apparatus, heat is always problem in electrophoresis.
- Some frowning or smiling is unavoidable, heat transfer is better at the rim than in the middle of the gel. It just should be a gentle, even curve, not a slalom parkour.
If you add ice to cool down your running buffer while running the gel I would recommend throwing in a stir bar and putting your apparatus on a magnetic stir pad to keep the temperature homogeneous throughout the buffer. If you have access to a cold room it might be better to run your gel in a cold room to prevent from diluting your buffer.
I will suggest to look into two factors to troubleshoot this problem:
(1) Decrease the voltage to 150-160 V because more the voltage, it will generate more heat, so the heat may not be distributed homogeneously through the acrylamide gel, thus gives result to 'frowning' bands. As you were fine with 200 V in the past, so you can start running the electrophoresis at 120 V, when the migration will cover the whole stacking gel and will come to the resolving gel, you can increase your system voltage.
(2) Check the concentration and pH of the TRIS-HCl buffer of the acrylamide gels. It should be freshly prepared and sterilized because the deterioration of buffer concentration will hamper the ionic potential in the system. It gives the same result as 'smiley' band in case of agarose gel electrophoresis. Also, pH is important in gel formation, as it makes the migration of the negatively charged molecules (chloride, proteins and glycine) in proper sequence.
Best wishes.
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