Hi guys,
Hope everything is going well with you. So I am encountering an issue, in which select (usually one or two) stacking gel wells rupture following loading the samples (usually containing 10 micro gms total protein), and the wells start leaking the samples. I am using 12% gels by the way. Would increasing the volume of TEMED, and APS, while preparing the stacking gel solve this issue. Also it is worth noting that the sample leakage doesn't always happen right away, sometimes it occurs minutes following the loading. Many thanks
HI Moe
If I understand your question well, during loading samples, the wells ruptured?
If your PAGE quality and well shapes are ok, use insulin syringe instead of 1-10 sampler for loading part.
Good luck
Always use Bio-rad reagents for gel preparations or any well renowned company reagents that are reliable. Make sure to add freshly prepared APS and TEMED during preparation of monomer solution. If it is one or more days old then increase the volume of APS. Moreover, during loading the sample try not to dig the wells and avoid longer time after loading the sample as it starts to diffuse along the wells. I hope this will help you.
GOOD LUCK...............
First check for the reagents used and make sure if the gel is solidifying properly. If you are sure that your stacking is solidifying properly, then the leaking of sample could be only an issue of loading method. If you are using a tip try not to press it hard in the well, which may lead to separation of the two plates more than the thickness of gel. This may lead to leaking.
hi. probably it is personal mistake. because TEMED use in very little and you must be fast. so its possible you dont add TEMED very well.
Moe,the real issue here is the amount of TEMED you used to polymerise the stack-it happens when the stack polymerises too fast which is what we do by adding excess of APS and TEMED to aid in good well formation.
The only solution is optimise the ratio of APS TEMED for stacking gel and then remove the combs immediately,rinse the wells with water or buffer using a syringe as suggested above and ensure no strands of polymerised material -remain int he wells
This is from an experience of running 100s of PAGE gels-
Dear Moe,
My suggestion would be to not to use too much TEMED as it would just shoot up the polymerization rate and thereby would make your gel fragile and more prone to break or rupture.
For troubleshooting, if your lab members are also having the same issue, please do check your reagents, especially the acrylamide. Prepare a fresh stock of acrylamide mix, APS and make a gel and check if it is gone or not. If your are the one only facing problem, then only suggestion is to practice more because some people, mostly during their initial days, end up scratching the well walls and surrounding during loading which ends up in leakage.
If reagent is an issue, do ask for small amounts from other labs before ordering a new one.
Please excuse me if it sounds in-appropriate or in-applicable to you, my guess is that it is more to do with your loading procedure. Please be patient and steady while doing it. Provide a support to both of your elbows while loading the sample with one hand holding the pipette and another one supporting it to keep it steady.
Wish it helps,
Best wishes.
Hi Moe,
Would increasing the volume of TEMED, and APS?
My answer is yes from my experience increasing those agents would solve it.
Good luck
Mahdi
One or two things come to mind here.
1. Your wells have un-polymerised acrylamide in them - pleases make sure you have flushed the wells with buffer before use. Stacking gels are usually 3-5% acrylamide.
2. There has been some mechanical damage to the well walls during gel preparation. this can be checked by adding diluted 1:10 sample buffer alone to wells to observe leakage.
3. You are using too much TEMED and APS, thus shrinking the gel around the wells during polymerisation. This might cause the gel to shrink from the plates, causing leakage.
Hope this helps
I too faced the same problem. I changed my acrylamide solution and opened a new vial of APS. It's resolved.
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