Hi all,
Recently I've been having issues with my SDS-PAGE gels not running properly. The samples don't run in a straight line as they should, but instead the lanes in the middle of the gel migrate way behind the lanes at the edges of the gel, leading to a dye front in the shape of a frowny-face. In addition, the lanes in the middle of the gel compress and become narrower, while the lanes at the edges of the gel expand and become much wider than the width of a lane. Does anyone know what might be going wrong? The gel rig itself is not the issue since another lab member has been using it for western blots without incident. I've been running my gels at a constant 180 V and have made fresh gel solutions and fresh running buffer containing the appropriate amount of SDS. I've been heating my protein samples for 10 minutes at 65 C before running, and this has been happening both with my purified samples (in 20 mM sodium phosphate, 0.5 M NaCl, and 500 mM imidazole) and with samples I have desalted using a Zeba spin desalting column. The protein itself is a positively-charged DNA-binding kinesin around 20 kDa. Could it be that I need to adjust the pH of my gel solutions to better suit my protein's charge?
Any suggestions would be greatly appreciated!
Thank you!
180V may be too high. How hot does that gel get? Also, are you sure there are no air bubbles below the gel in the tank? That would block the current and give you weird migration. You pour your own gel, right? Is there any chance the plates are coming apart a bit? If you're not clamping the plates together over the spacers, you might be compressing the plates too much, and then after the gel is polymerized and you take off the clamp, the gel is not uniform in thickness. And, yes, as suggested by Ahmed H., be sure you mix your gel well (but I might suggest degassing the solutions before adding the APS and TEMED).
I can't really see the gel you posted but it doesn't look good. More suggestions. 1) Run a gel using your labmate's conditions and confirm that you can run a good looking gel on that apparatus. 2) pre-run your gel before loading, if you aren't doing that already. 3) run your gel with much lower voltage and see how it looks.
A really long time ago, back in the days of sequencing gels, I learned the hard way that you can run an acrylamide gel too hot - they don't melt, exactly, but they deform and don't run well. So if you have hot spots (you can feel the gel plate...with the power off), that's not good. If you need to run the gel at high voltage, add lots of buffer so the gel is covered as much as possible - it will act as a heat sink. But if the weirdness is happening at the part of the gel that isn't covered with buffer, maybe heat is your problem.
I'm not sure if you need to adjust the pH, but the pH of the buffer will change during the run. Again, back in the "dark ages", I ran some gels with recirculated buffer (between the top and bottom tanks), with the thinking that the gel looked better because we weren't making a pH gradient during the run (I don't really know if that explanation is true, but I've checked the tank buffer pH before and after and it can change).
Last question - are the weird lanes associated with a particular type (high salt/desalted?) of sample? If there is a correlation, that might be the hint you need to figure out how to fix this.
Thank you, I think the problem here within the preparation of gel itself
1. make sure that you add the solidifying agent (TMEM) and shake the whole resolving 10% solution vigorously and transfer it spontaneously to your gel glass frame and make sure you add isopropranolol not water, water is a main component of your gel so it's not preferred.
2. use 10% resolving solution for 20 KDa, and run your samples at voltage level up to 130
because 180 is so high seemed to pull your samples down and this is not good for enhanced running.
180V may be too high. How hot does that gel get? Also, are you sure there are no air bubbles below the gel in the tank? That would block the current and give you weird migration. You pour your own gel, right? Is there any chance the plates are coming apart a bit? If you're not clamping the plates together over the spacers, you might be compressing the plates too much, and then after the gel is polymerized and you take off the clamp, the gel is not uniform in thickness. And, yes, as suggested by Ahmed H., be sure you mix your gel well (but I might suggest degassing the solutions before adding the APS and TEMED).
I can't really see the gel you posted but it doesn't look good. More suggestions. 1) Run a gel using your labmate's conditions and confirm that you can run a good looking gel on that apparatus. 2) pre-run your gel before loading, if you aren't doing that already. 3) run your gel with much lower voltage and see how it looks.
A really long time ago, back in the days of sequencing gels, I learned the hard way that you can run an acrylamide gel too hot - they don't melt, exactly, but they deform and don't run well. So if you have hot spots (you can feel the gel plate...with the power off), that's not good. If you need to run the gel at high voltage, add lots of buffer so the gel is covered as much as possible - it will act as a heat sink. But if the weirdness is happening at the part of the gel that isn't covered with buffer, maybe heat is your problem.
I'm not sure if you need to adjust the pH, but the pH of the buffer will change during the run. Again, back in the "dark ages", I ran some gels with recirculated buffer (between the top and bottom tanks), with the thinking that the gel looked better because we weren't making a pH gradient during the run (I don't really know if that explanation is true, but I've checked the tank buffer pH before and after and it can change).
Last question - are the weird lanes associated with a particular type (high salt/desalted?) of sample? If there is a correlation, that might be the hint you need to figure out how to fix this.
Thank you both for the quick replies.
I have been pouring my own gels and clamping them together, so I think I can safely eliminate non-uniform gel thickness. I'm not sure about bubbles- that's definitely something I'll check for next time along with shaking my gel solution more vigorously and running at a lower voltage/prerunning before loading samples.
I've been running the gels in a cold room set at 4 C, so I doubt overheating would be the issue? Unfortunately, the weird lanes don't seem to be confined to any one type of sample. It's happened with nuclear extracts, concentrated samples with high salt, and desalted samples.
I usually have a similar problem of the dye front running unevenly when I haven't put enough running buffer in the tank. Maybe that's your problem as well?
Ah, yes, Shruti has a good point. If your buffer level sinks during the run, you'll get weirdness.
To eliminate bubbles from beneath the gel, I used to use a bent syringe needle. Suck up some buffer from the tank and blow out any bubbles hiding under the gel.
Hi
Two things that may cause this
1- Temp (4 C), I don't know the reason behind running SDS (denatured gel) at 4C because SDS precipitate at low temp. if to be carried at 4C (but why?) then replace SDS with lithium dodecyl sulfate.
2- I agree with Shruti, it's good practical to fill the clamped gel tank (not gel rig tank) with buffer or even Milli Q H2O (in order to check for leaks 3-5 min) before place it in gel rig.
Good Luck
Thank you so much! I filled the tank up with more running buffer and ran at 100 V, and everything is running beautifully.
This is most likely due to either of two situations below:
Either buffer is level is decreasing in the gel cassette due to a minor leak. Check that. If buffer leakage is not there you may want to try a fresh SDS running buffer.
There my be bubbles in some parts of the gel (some visible in the lower part). Remove bubbles before loading samples and running the gel.
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