For a certain antibody staining I am doing a RNAse treatment, but the protocols usually mention doing this treatment in Tris-HCl buffers containing NaCl and usually a chelator like EDTA or sodium citrate. Is there a reason for this? Since I use PBS for all steps during IFC, I was just using PBS for RNase treatment but I wonder if this affects the enzyme efficiency despite RNase being a very stable enzyme.
It depends, for RNAse A PBS is okay, you can use it as a buffer for digestion for other RNases IF, III, or H I used them wither their provided buffer.
Hope it helps.
Hello Shravasti Misra
It is essential to comprehend how these buffers interact with metal ions and other biological materials like proteins. For instance, in enzymatic reaction the enzyme requires metal ions for its activity which may be inhibited if the buffer that is being used forms an insoluble complex with those metal ions. Phosphates form insoluble salts with bivalent metals and precipitate.
Therefore, Tris buffers are preferred to PBS because Tris buffers are inert in many enzymatic systems (no interactions with other components). As rightly mentioned by Mamadou Amadou Diallo , it depends upon the type of RNAse you will be using for RNAse treatment.
For example, the addition of Ca2+ is required for RNase I to fully degrade the substrates and RNase E requires a divalent metal ion like Mg2+/Mn2+ for its activity. On the other hand, RNase A does not require metal ions or cofactors for its activity.
Best.
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