I just wonder even after following al the protocols and preparing the stacking gel, it will only form a semi-solid gel. And refuse to solidify like the loading gel why?
Hello,
I imagine you are trying to run SDS-PAGE. Please find attached the protocol we use in our lab for our gel prep. You might give this a try. I usually make my own gel and have never had any issues (at least with polymerization). However, ensure your APS and TEMED are of electrophoresis grade and the Tris buffer is at the appropriate pH indicated on the protocol.
Cheers.
What do you mean by "semi-solid gel"? A stacking gel for a protein acrylamide gel has a lower percentage of acrylamide (and a different buffer). Good wells should form and you should be able to load it just fine, and when running the samples will "tighten up", or stack, and run quickly through the stacking gel before hitting the running part of the gel. When you disassemble the gel, however, you may notice that the stacking gel isn't as solid because it has a lower % acrylamide. The stacking gel is more fragile, and I used to cut it off before imaging the gel. If that is what you mean, it is normal. Compare your percent acrylamide in stacking and running gel and see if it makes sense.
If, however, the stacking gel isn't polymerizing well, and wells aren't forming, something is wrong. Give us more information please.
I think you should first check the pH of both buffers and try APS from new bottle and not from that which has been opened few years ago. I think it works.
I really appreciate your answers. Thank you so much. I have done it and it worked. @Mr Ojo. I am greatful. But I don't know if you have notes on the following topic, I have a presentation on them and I trust it's your aspect.
1...Enzymatic Browning
2...GMO in food: Biochemistry and application
3....Food enzymes and their applications in food industry. Thanks @mr oklahoma
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