We are planning to carry out a protein cleavage reaction, we would like to see if the cleavage reaction occurs in acidic pH (5.5). The protein has been purified, concentrated and saved in pH 8 buffer (500mM NaCl, 10mM Tris). what is best way to bring the pH to 5.5 to observe the cleavage reaction in the protein.
Your suggestions will be appreciated.
There are at least 4 convenient ways.
(1) Dialysis against the pH 5.5 buffer.
(2) Gel filtration using a desalting column equilibrated with the pH 5.5 buffer.
(3) Diafiltration. Use a centrifugal ultrafilter to concentrate the protein, then dilute it with the pH 5.5 buffer. Repeat a few times until nearly all the pH 8 buffer has been replaced with pH 5.5 buffer.
(4) Add a concentrated low-pH buffer directly to the sample to overcome the buffering of the Tris and bring the pH to 5.5. Do not add acid to adjust the pH of the protein solution. Some experimentation will be needed (without protein) to get the final pH just right.
Thanks Adam for the valuable suggestions. As the proteins were saved at very low volumes around 20ul aliquots. Option 4 looks to be the ideal way.
Just try first, how much you will need to add (you may scale it up to 20 or 40 ml so you can easily measure with pH meter).
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