Hello,
I have ATP from Sigma as a salt and want to prepare it as a solution (probably 100mM stock) for cell culture work in activating inflammasomes. I have a few questions in the handling of this.
1. I am thinking of pH-adjusting the solution to ~7 once I dissolve the salt. Will I need to keep the solution on ice while I adjust the pH to prevent ATP degradation? Can I dissolve the ATP in PBS instead of water and will that affect the pH/do I still need to adjust the pH?
2. The pH meter we have in our lab is outside the hood so I am thinking to syringe filter the ATP solution to sterilize it. Does it matter which membrane material I use as long as it is 0.2 um pore size?
3. After thawing an aliquot from -20, is it only good for one use? Can I keep it at 4 degrees and if so for how long? Alternatively will it last through a few more freeze thaw cycles?
Thank you!
Jieun Stella Lee
This instruction sheet answers most of your question. ATP is quite stable, and 4ºC is OK. Any membrane filter of 0.22 um pore size will be ok. I am not sure is you can get 100 mM. This is quite high concentration, and the solubility of the salt depends on the number of sodiums in the phosphate groups (at to four). At pH 7, I think that 4 sodiums will be needed.
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/a3147pis.pdf
Hey Jieun :)
I prepared my ATP directly in culture medium (usually 50 mM) and used it fresh, within an hour or so of preparing. ATP loves to hydrolyze.
If you do prepare it in PBS or the like, pH it with litmus paper if you can and that allows you do avoid needing to filter it. If you do end up filtering it, that's okay too. Store your aliquots at -80C and consider them single-use.
Good luck!
Edit: This is an important paper to consider when performing these experimernts:Article ATP-Induced IL-1β Specific Secretion: True Under Stringent Conditions
Hi Jordan!
I went ahead and did the syringe filtration and aliquoting before I saw your answer. I am doing condition optimization experiments right now so I will use the aliquots I made for those but I will definitely keep your advice in mind for the readouts and for the next time I prepare ATP.
Thank you for your help and the paper!
Under the right conditions, i.e. absence of ATP degrading enzymes and neutral pH, ATP is much more stable than you believe.
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