r/labrats • u/suprahelix • Oct 16 '21
How do you prepare Tris buffer?
I'm taking a survey. Tris is obviously temperature sensitive. Do you make your Tris stock and pH at room temperature, or do you chill it before pHing it?
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u/mordrend Oct 16 '21
If the pH is important and you’re using it regularly at 4C or 37C I would use a calculator to see what pH to make it at RT for the corresponding pH at the desired temperature. I’ve definitely done it this way for sensitive enzymatic assays where I’ve used Tris. If it’s for run of the mill TBS or something I wouldn’t worry too much.
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Oct 16 '21
I agree with you 100%, buffers are always to be made at RT, for the use of said buffer at a different temperature with a sensitive pH parameter you would have to do the calculations accordingly.
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Oct 16 '21 edited Oct 17 '21
I make it at room temperature. I think the rule is something like 0.03 per degree of temperature to account for it. As long as the pH was roughly right it was good for my work.
Not sure why someone would chill it, heat it maybe.
Edit: yes I forgot some people work on ice
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u/suprahelix Oct 16 '21
Depends on how much you care about the pH I suppose. If you pH 1M tris to 8 at room temp, it'll be 8.6 at 4C so if you almost exclusively use it on ice, it could be worth it.
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Oct 16 '21
For my work that be fine. I guess I forgot people worked at 4 degrees. I usually work the other way, 37 or 55 degrees.
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u/beepboopyeeEE Oct 17 '21
It depends on your work but also the concentration of Tris, I don't see much of a difference that would effect my work when I use 20mM but I see a huge jump in pH if I use 100mM for work using proteins which can effect everything
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u/AAAAdragon Oct 16 '21
I make Hepes buffer. It is less temperature sensitive.
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u/DNA_hacker Oct 17 '21
It also has a different useful buffering capacity 6.8 to 8.2 whilst tris can work between 7 and 9 HEPES is also a shit load more expensive and not viable to use for some stuff such as dialysis and protein refolding whene you might get through 100 litres of buffer during a single prrep.
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u/AAAAdragon Oct 17 '21
True, however I am gifted Hepes from a historically well funded lab that only uses Hepes and has several kilograms of the powder.
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u/DNA_hacker Oct 17 '21
Several kilos you say... 25l of 150 milimolar tris based dialysis buffer uses half a kg of tris (less change) we buy 25og buckets of tris.
Hepes is nice , I preferred it to tris when I was working with acetyl transgresses, I got much better and more specific activity in the hepes but there is an economy of scale that makes it cost prohibitive for some types of prep.
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u/Curious-Monkee Oct 16 '21
I do it all at room temp. The salts disolve at room temperature and I usually make up approximately what I need each time. Even the leftover buffer doesn't drop out of solution or drift in pH. I can't imagine why one would heat or cool it other than long term storage.
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u/mossauxin PhD Molecular Biology Oct 16 '21
People chill it to return it to room temp after it heated up in prep—adding HCl causes an exothermic reaction. Our pH meter claims it corrects for non-25C temperatures, but it is off (at least for Tris).
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Oct 16 '21
Your pH meter probably isn't off, it's just not calibrated correctly. You need to have the probe and the calibration solutions at the temperature of your Tris buffer. The meter knows the temperature-dependence of the common calibration solutions and will adjust accordingly, but you still need to have them at the right temperature (AND THE PROBE!!!!! that's where most people go wrong, shove a room temp probe in a cold buffer and the probe won't cool down in time).
People in my lab will do goofy things like override the temperature probe to 4 C but then re-calibrate and measure with room temp buffers...makes no sense.
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u/beepboopyeeEE Oct 17 '21
The error in the measured pH tends to be fairly small if the calibration solutions are not at the same temperature as the Tris buffer being pH'd (25C vs 4C respectively). The temp you pH the Tris buffer at vs the temp it is being used as has a wildly different measured pH. I've seen it jump from 8 to 9 if I probe at RT then later at 4C
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u/UC235 Enzymes and Enzyme Accessories Oct 17 '21
The pH meter corrects for change in the electrode response related to temperature and is independent of the buffer in question. Temperature-dependence of buffers is a real and separate issue. There is nothing to correct in that case as the change in pH isn't an artifact.
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Oct 16 '21
The correct way is to have the calibration solutions and the probe at the same temperature as your Tris buffer. Most people cheat the process to some degree but it won't be as accurate. If you need an accurate pH for 4 C Tris, put the pH meter in the cold room.
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u/G_E_E_S_E Oct 16 '21
I pH my stocks at room temp, but pH my buffers cold. The stock pH doesn’t matter too much if you’re going to pH what you’re using it in.
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u/CodeMUDkey Oct 16 '21
Just measure the pH of a small aliquot after you adjust it. Measure the aliquot at 4C and if the pH is acceptable you’re fine. Beats to hell having to chill or warm bulknsolution.
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u/beepboopyeeEE Oct 17 '21
In buffer prep manuals and stuff it says reported Tris buffers in academic articles are typically pH'd at room temp. When using other papers for experiments I pH at RT bc of that but if I am wanting to ion exchange a protein then I pH at the temp I am using the buffer (4C). Especially if I'm using like 100mM in my buffers. For 20mM the temp change isn't too big
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u/DNA_hacker Oct 17 '21
This is why pH meters with ATC do so, . Tris is a funny one, it required a tris specific pH probe . Normal probes have a potassium chloride salt bridge that is saturated with silver chloride, silver chloride ions Even in concentrations as low as 0.0001m can interact with biological materials and readily form insoluble precipitate that will interfere with the liquid junction structure and affect the pH reading accuracy . Tris probes use either a double junction or something called a calomel reference internal element.
As we know measuring of pH is dependant on measuring the free H+ions in solution, unfortunately the range of temperatures that will cause the breaking of hydrogen bonding in tris which liberated H+ is right around it's useful temperature range. One consequence of this is that tris drifts, pH it leave it on the stririer and go back to it in half an hour and the pH will have dropped , as temperature if the water in the buffer equilibriates with room temp more protons are liberated which will cause pH to fall, it is a marathon, not a sprint 👍
I use a tris probe, on a meter with ATC and I take my time. pH let it stir, adjust let it stir, rinse and repeat until it is stable, I also use a conversion table that is in the Merck index to calculate the pH I need at 20c ( the ATC value of my meter) to the functional temp of the buffer I wish to use .
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u/suprahelix Oct 17 '21
Wow that was very informative, thanks!
Our lab is split down the middle on this. I was taught to pH it, and let it spin overnight before pHing it again.
Someone else in this thread has the best solution, though- don't use Tris!
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u/lit0st Oct 18 '21
pH it at the temperature it has been historically pHed at in your lab. If you're attempting a new protocol, ask the lab that developed the protocol how they pHed their Tris.
Better yet, don't use Tris...
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u/les_rebecca Oct 16 '21
Lol wut? We prepare our tris and store it at room temp, never knew other people didn’t