The carbonate buffer is provided with the ATTO643 labeling kit because the reaction of NHS-esters with lysines is most effective at around pH 8.3. At pH 7.4, most lysines are protonated and not available for the reaction. Although (de-)protonation is an equilibrium reaction, allowing all free lysines to eventually be labeled at pH 7.4, the process will be slower and less efficient. Additionally, NHS-esters slowly hydrolyze in aqueous buffers, so a longer reaction time results in more NHS-ester decay.
If the protein tolerates high pH but not the carbonate buffer, another buffer can be used and adjusted to pH 8.3. A HEPES buffer has been successfully used for this purpose, as it maintains sufficient buffer capacity at pH >8. PBS can be adjusted to pH 8.3, but this is outside its buffer capacity, requiring careful monitoring of pH fluctuations. Refer to the buffer chart in Figure 3: https://www.sigmaaldrich.com/technical-documents/technical-article/protein-biology/gel-electrophoresis/reproducibility-with-biological-buffers. Avoid buffers containing primary amines, such as TRIS, as they will compete with lysines and reduce reaction yield.
A pH of 8.0 might be sufficient with otherwise identical reaction conditions and is tolerated by many proteins.
If using PBS at pH 7.4, the reaction will occur more slowly. Instead of the one-hour reaction time in the protocol, label for three hours or overnight. Increasing the dye concentration is also an option. The kit provides enough ATTO643-NHS to use twice the concentration or more. Conduct a test reaction, measure the degree of labeling (DOL), and adjust reaction time and/or dye concentration if necessary. A starting point is a 3-hour reaction time at room temperature with 1.5x the dye amount recommended in the protocol.
