Buffer selection

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Thilllai Punitha
Thilllai Punitha's picture
Buffer selection

Dear all,I would like to ask regarding Tris buffer. As i'm following my literature review, they used Tris-HCl for dissolving pellet form crude enzyme and also for dialysis.In all the previous studies they used 50mM Tris-HCl at ph 9.But i'm not sure why the buffer doesn't work for me? Extracted crude i have tried with acitivtiy analysis, the enzyme give the reaction but after i add this buffer it seems like not activeI do my steps like this after extraction:1) Tris-HCl buffer at pH 9 prepared in room temperature about 25C. Then stored in 5c chiller.2) Pellet will be dissolved in this buffer (about 4 ml buffer + 2ml pellet) and i will stored in -20C.3) I will do dialysis in the in the same buffer Tris-HCl (pH 9) at 4C in the cooling chamber3) Then after when i'm about to use the enzyme , the activity is not there anymore as i felt like th enzyme denatured.Any suggestionThanks

Fraser Moss
Fraser Moss's picture
Temperature dependence of Tris buffered solutions

I read that you setting the pH of your Tris buffer to 9.0 at room temperature, but then doing the assay at 4 degrees and storing at -20 degrees.  Tris buffer is very temperature sensitive.pKa changes +0.028 pH units for each degree Celsius when temperature decreases, or -0.028 pH unites when temperature increases.pH = pKa log [A-]/[HA]If room temperature is 25 C but you'll perform the assay at it at 4 C, you will have a decrease of +0.028 x 21 = +0.588 pH units   - so your buffer will probably be ~pH 9.6, not pH 9.0. At -20 C this could go as high as pH 10.26.At these pH's you could be danaturing or killing the activity of the protein.You should set the pH of the buffer at the temperature that you are going to perform the assay, or caluclate the pH at room temperature that will give you the correct pH at the working tempartaure.see.https://www.neb.com/tools-and-resources/usage-guidelines/ph-vs-temperature-for-tris-bufferhttps://www.applichem.com/fileadmin/Broschueren/BioBuffer.pdf