I bought 10mg RNAse from Sigma, which comes in a drop of 50% Glycerol and Tris - HCl. I want to add 1mL solution to bring it to a nice working concentration of 10mg/mL. Now we discuss in the Lab in what dilution. I thought stay with 50% Glycerol as otherwise the enzyme gets damaged when frozen and thawed all the time, others say dilute it immediately in PBS as I will use it at the end in PBS.... can anybody help ?
Thanks in advance
Hi there,
RNAse has the advantage to be very robust so it's up to you. You may even boil it before use to prevent any DNAse contamination. Personally I would go for PBS.
Recombinant RNase is very robust, and most will survive refreezing, although some will always die. I highly recommend not ever using phosphate to store proteins. Unlike organic buffers such as Tris, MOPS, etc, phosphate buffers cause local heating around the protein during freezing and thawing, which damages the protein. If you aren't going to use all 10 mg on the same day, I recommend diluting into your desired volume (ice cold after thawing the current solution on ice) with 10 - 50 % glycerol in an organic buffer, and then aliquoting into smaller volumes that you will use in a single experiment (this goes for almost all proteins really). Flash freeze them in liquid nitrogen and then store them at -80 C.
Having said that, the RNase that is present in plasmid extraction kits is stored in the fridge for months on end and still works fine. So it depends on whether you specifically need very high activity from your RNase. Remember that proteins and buffers stored in the fridge for extended periods of time must be kept sterile at all times.
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