Does anyone know why we are having problems with the buffer of PB at pH 5.0?
After different tests, we ended up thinking that the problem with some stainings in our lab is the buffer used of PB at pH 5.0. This is how we make it:
We mix:
- 14.40ml of destilled water + 0.86g sodium phosphate
- 75,60ml of destilled water + 5.37g disodium phosphate
Then we mix them and add chloridric acid to adjust the pH at 5.0.
Finally, we mix the solution with 360ml of destilled water and 3.06g of sodium acetate.
Does anyone know what's wrong with this protocol? When we use this buffer as a copper stock solution we observe the two-phase situation, which can be seen in the pictures attached. We have already checked that the copper sulphate is okay, so it seems that the problem comes from the buffer.
Thanks in advance!
Hi Laia Vila . You might have precipitation of copper phosphate. Check solubility tables.
This protocol seems to be strange to me. Where did you get it from? What is the purpose? A buffer of pH 5.0 is usually obtained by a pure acetate buffer, without any phosphate.
https://www.aatbio.com/resources/buffer-preparations-and-recipes/acetate-buffer-ph-5
Hi Michael G. Weller , we use this buffer to incubate brain slides for acetylcholinesterase staining. We've been using it for a long time, but this is the first time that we're having problems with it. Since the buffer gets these precipitates, my tissue staining ends up with precipitates (you can see them in my last question published in researchgate). I'll check on September with the lab technicians about your suggestion. This seems quite new to me!
"Phosphate buffer at pH 5" is an oxymoron. It's not a buffer at pH 5. It has an extremely low buffer capacity pH 5. "We've been using it for a long time" At what pH?
Yurii V Geletii We've been using this buffer and the same one at ph 7.4 for acetylcholinesterase staining for a year (I attach protocol). Since we've recently found precipitates in our brain tissue, we've been studying the reasons that could cause it. Finally, it seems that the buffer at ph 5.0 used could be wrong, because after adding copper sulphate we observe this two-phase solution. The surprising thing is that we've used this protocol before successfully, but the last few times has ended like this. Do you know what could be wrong with it? Thanks in advance.
The attached i slides are not informative. What is the concentration of pH 5 buffer? An addition of sodium acetate may shift pH to higher values. Did you check the pH after each step?
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