RNase A preciptitation

David F. Spencer dspencer at is.dal.ca
Wed Jun 21 10:42:41 EST 2000


in article 8h8uij$vao$1 at mercury.cc.uottawa.ca, Neil McKenna at
nmckenna at ottawa.com wrote on 6/2/00 3:24 PM:

> We recently prepared come RNase A as a 100 ug/mL solution as described in
> Sambrook, Fritsch and Maniatis  -- we made a solution at pH=5.2, boiled it,
> then adjusted pH to 7.4. We had been using it for several weeks and found it
> was degrading our DNA when used at high concentrations, so we decided to
> leave it overnight at 65 C. Unfortunately, when we came in this morning, it
> was all precipitated, and we cannot seem to resolubilize it.
> 
> Is there any way to resolubilize RNase A without having to dilute it?
> 
> Thanks
> Neil McKenna

By this point I would assume you've solved the problem one way or the other.
However, I think you've made the RNase A preparation protocol far more
complicated than it need be.

We have used Sigma's RNAse A solution (cat. R 5250) for many years with no
problems. The stock is around 0.75-1 unit per microlitre so I use 0.5 to 1
microlitres to treat 100 microlitres solution (this is almost certainly
overkill; the stock protein concentration is about 1 mg/ml). We kill any
DNases beforehand by aliquoting about 100 to 500 microlitres from the parent
bottle (of about 100 ml) into a screw cap microcentrifuge tube, placing it
in a 90 C bath for 15 to 30 minutes, then take the tube out and set it in a
rack to cool to RT at its own pace. I then store it at -20 C. No dilutions
or pH titrations.

Leaving the solution at 65 C overnight probably wouldn't do anything to the
RNase A (it's fairly stable and still functional at that temperature)
although the protein would probably slowly undergo oxidation that would
damage the sulphydryl groups and that would kill the enzyme. However, you
must remember that probably no RNase A is pure and for example some of
Sigma's are as little as 60% pure. That means that there are other proteins
in the RNase A prep and they may very well irreversibly denature and
precipitate with prolonged 65 C treatment. That is not unlike the standard
method of purifying Taq polymerase, which is stable at 70 C for prolonged
exposure whereas other E. coli proteins denature, precipitate and can be
spun out. So even after the 65 C the supernatant could very well have had
useable RNase A. You don't mention whether you checked it.

Dave

==========================
David F. Spencer, PhD
Dept. of Biochemistry and Molecular Biology
Dalhousie University
Halifax, Nova Scotia
Canada

dspencer at is.dal.ca
==========================







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