How to Avoid a Leaky SDS-PAGE gel
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- čas přidán 17. 07. 2024
- Because it's happened to all of us -- despite following the protocol, the gel leaks. What did you do wrong? Usually there's just a seal problem, and Dan shows some tricks that you can use to avoid a leaky gel.
Got your own trick? Leave a comment!
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14 years later and you're still saving lives...just had 3 leaky gel attempts and this video saved my ass
Sometimes the pen or parafilm trick might not work because the seal is still not tight enough. This is usually due to tiny cracks at the edge of your glass plate, or a worn out rubber sealing pad on the bottom. If you read through the comments below, there are a number of other tricks that you can also try such as: parafilm around the three edges of your glass plates; use petroleum jelly to seal the bottom of your plates; or use agarose to seal the bottom of your plates. Good luck!
THANKS!!! I had my gels leak on me today... 2x. I figured the seal was worn down.. I can't wait to try these tricks!
You can add a little amount of resolving gel with higher percentage of TEMED to prevent leaky and after the thin layer of the bottom becom solid , you can add standard resolving gel
WoW Thank you so much. The parafilm trick works like a charm!
Yeah thanks for this! I will try it. I just spent 1 hour repeating it! Urr! Thanks
@alabonneheure1 Make sure you are using fresh ammonium persulphate solution - if it's old, the gel will not polymerize (or will polymerize much slowly), increasing the chances of leaking.
Also, @parugoelbiologist just replied about the agarose trick: add a 4-5% agarose solution to the foam, then put the glass plates on top and let it solidify. Once solid, continue with resolving as usual. Good luck!
I had leakings and I have identified why i had them :) Thanks !
@sakaewan Thanks for the trick! Right, the pen trick is limited to this apparatus, but petroleum jelly should work for all casting sets. Thanks =)
You can also seal the sides, bottom front and back of plates and corners with 1% agarose !!
@born2strangesights cool! never thought about a microfuge tube before, but thanks for the tip! i'll try it sometime =)
you can use agarose gel to stop it from leaking
Pre-cast gels are where it's at!
@parugoelbiologist Thanks for sharing this! I've never used the agarose trick but will give it a try =)
@beeryya interesting! thanks for sharing =) could you provide more details about you use that? do you add the agarose gel to the foam underneath the plates, or do you put it in between the glass plates before the resolving gel?
when i try to make a gel, it's always leaks one side (of casting frame). I'm unable to fix this. Can you please help me regarding this
Nice
You can also load a double amount of TEMED, so the polymerization will go faster, there will be no time to have any leak😉
Double TEMED only? Not APS?
Hello,
I try to make zymography but have a serious problem with the result. I don't know where is our mistake. The bands after electrophoresis is very ugly and irregular. If i make a video clip tommorow can you help me if the mistake is visible?
Best regards
I tried these two tricks, but like someone else on here, I still had leaks. I also tried petroleum jelly, but this is somewhat messy as it leaves jelly on your casting stand and casting frames.
What worked best for me was resting the casting frame HORIZONTALLY after pouring the gel. I simply use a tube rack that is about 1in high on which to rest the casting frame on its back. Once gravity was (mostly) taken out of the picture, I have perfect gels every time, even when using old, slightly cracked plates and even without the use of parafilm, pen, jelly, etc.
Do you add/overlay water on the top of resolving gel in your method?
I tried your method by casting frame HORIZONTALLY after pour resolving gel and water-overlay. It is no leakage at all, but the gel wasn't polymerized.
Asa Wu Asa, 99% of the time a continuous gel is sufficient for me so I really don't use the water overlay method. It might be that when you lay it horizontally, the water is mixing with the monomer solution, interfering with the polymerizing step. That's interesting and certainly something I didn't think of when I wrote my comments above.
I should say that I really meant ALMOST horizontally. The point was the closer the angle between the plane of the gel and the bench is to 0 degrees the better, from a leakage perspective. However, I've noticed I can increase the angle to about 45 degrees while still obtaining minimal leakage. Obviously this depends on the condition of your plates, seals and gel reagents as well.
I'd suggest adding the monomer solution, placing the gel at a 15-30 degree angle, THEN slowly adding the water overlay. You can play around with your system to figure out the best angle you need to minimize leakage while preventing the water from mixing with your monomer solution. Good luck!
Michael Anaya Thank you for your reply. I will try your suggestion and reply the result.
I used gelatin dissolved in water, i let it dry and added the solution
Thanks for your solution! Which percentage of gelatin did you use?