I currently have significant doubts regarding the pH of the protein purification buffer. When purifying proteins, do I need to calibrate the buffer pH at the purification temperature? For example, if the protein purification system is placed at room temperature, should I adjust the pH of the purification buffer at room temperature? The protein purification is performed at room temperature, followed by concentration at 4°C, and storage at -80°C by freezing.

For purifying E. coli DnaA protein, with a predicted isoelectric point (pI) of 8.77, if I purify at room temperature, can I simply adjust the purification buffer pH to 7.5 at room temperature? The purification buffer is typically 50 mM Tris-HCl. When the protein is stored at 4°C, the buffer pH rises to approximately 8.1, indicating that this buffer pH is acceptable.

Correspondingly, for a DnaA protein from another species with a predicted pI of 7.2, also purified at room temperature, can I adjust the purification buffer pH to 8.0 at room temperature? Additionally, when the protein is concentrated and stored at 4°C, it should not be affected because the buffer pH increases at lower temperature, moving away from the protein's pI of 7.2, thus not compromising the protein state.

Can I understand it this way?