Milu
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decomposition
what is the decomposition temperature of sodium pyrophosphate? Can i autoclave its solutions without fear of decomposition?
pyro
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vulture
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Römpp gives a melting point of 880C or 988C, so autoclavation should be no problem.
One shouldn't accept or resort to the mutilation of science to appease the mentally impaired.
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chemoleo
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Can't autoclave it!
No, the problem during autoclaving is that it is in solution.
In the lab we sometimes use it as phosphate buffer replacements (i.e. when phosphate itself is to be avoided).
A buffer containing pyrophosphate should be discarded after a few days at 4 deg C, for it then contains appreciable amounts of phosphate due to
hydrolysis (if phosphate is a problem, which is normally the case if one chooses to use pyrophosphate). Similarly, autoclaving will most definitely
hydrolyse it in a short time, so it is a no-no ....
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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Milu
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I am using the pyrophosphate for making the "stripping solution" for a Northern blot. Working with RNA samples are rather dicey and if i
cant autoclave the solution...then filter sterilisation would be the alternative. But then again one cant rule out contamination with RNAase(which is
a very hardy enzyme)...Would it be ok if i immediately use the prepared and autoclaved pyro solution?
pyro
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chemoleo
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Interesting! Can I ask what/where you are working on (U2U if you prefer)? I once worked on RNases, and RNase inhibitors, and it's been long
established in that lab that only certain reagents seem 100% RNase free (which is an issue if you are working with highly active enzymes, and highly
active inhibitors, whereby pico- or femto-molar concentrations matter). A specific supplier we used for that reason was CalBiochem. Sigma etc buffers
did have (surprisingly enough) minute RNA cleaving ability. We found that autoclaving did NOT destroy RNase activity 100% (which is a well-known
problem).
The answer to your question - NO, you can't use it immediately after autoclaving - 120 deg C will be more than enough to facilitate hydrolyis of
most of the pyrophosphate.
There are two options to go from here:
1. There are special protein filters called SepPak (or something like that) that bind any type of protein, thus riddening your buffer from residual
RNase activity. This we frequently used.
2. Another option is of course to use the RNase inhibitor (RI), which can be ordered from Promega. It is patented of course. The RNase inhibitor is
VERY effective, add a little and you won't ever have problems with the RNases again. Needless to say, you can't autoclave this, as RI will
be denatured.
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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kryss
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pyrophosphate hydrolysis
I would have thought that pyrophosphates would be reasonably stable when autoclaved as long as the solutions not acidified. One of the problems of
analysing for Total Phosphate is ensuring complete breakdown of the polyphosphates.
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chemoleo
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Yes, but as you say yourself, it's *complete* breakdown.
People who chose pyrophosphate normally have a good reason to, which implies that phosphate cannot be used. Small concentrations of phosphate may be
enough to screw up your experiment. And autoclaving will most definitley produce phosphate (I am not debating percentages here...).
As I said, in the lab we are advised to dump pyrophosphate buffers after a few days, even at 4 deg C.
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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