I am experiencing many problems in obtaining the correct pH of Tris-HCl solutions for acrylammide gels (pH8.8 and 6.8) due to the dependence of the pH on temperature. I prepared and brought the solutions to pH at room temperature but then I stored them at +4, and measuring the pH again I observed that it was completely different. how can I do? I can use the solutions at room temperature (I usually prepare gels at Rt but then I run the Western blot in ice or in cold room) or I have to pH on ice bucket? Someone has the same problems?
Thank you
This is always a pain. pH the Tris buffer at the temperature for use, or use a table like the one in the link. It is pretty close. https://www.neb.com/tools-and-resources/usage-guidelines/ph-vs-temperature-for-tris-buffer
You can just calibrate pH meter and prepare buffers at room temperature both 6.8 and 8.8 then you store them at room temperature or 4C it dosent matter you just use those buffers for gel, western blot- at room temperature or on ice. I store buffers at 4C, they work fine for months.
Unless you are running your electrophoresis at 4C, it does not matter. The pH will change upon cooling, but it will also change back to the pH 6.8 or 8.8 upon warming to room temperature.
You can run at 4C, you just have to change the pH for RT.
Calculate the change in pH over the temperature change using the delta pKa/delta T-value for Tris (-0.028pH/K) and then prepare the buffer accordingly.
These are the bands I obtain, both for the markers and the protein. Do you have any ideas about the reason? I thought it could be for the tris HCL buffers but I obtained the same from a precast gel... I have never had such a problem in my previous lab and the only differences were that I used precast solutions both for the gel and for the running and transfer buffers. Thanks
Have you tried running the gel in a different electrophoresis chamber?
At least some of it looks to me, as if you have an electrical leak somewhere.
However, it is also strange that the waviness in your bands is from the very top to the bottom of the gel. There could be something wrong with your stacking of the sample in the stacking gel.
Check or better just exchange your sample buffer, how old is it?
Do others using your sample buffer see the same problem?
Achim Recktenwald Hi Achim, thank you for the suggestion. The sample buffer is the only think I had not considered and it could be. i have tried to change everything else but still using the old sample buffer. I will try to prepare another one new. Thank you.
still thinking about it, but if it was for sample buffer, why also the marker is wavy? It could be not for the chamber but the power supply instead? Achim Recktenwald
Yes, on my way home yesterday, I was thinking the same :)
The power supply could be the cause. Yes, try changing it.
I would even run it from a different electricity socket in your lab.
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