The first step in choosing your lysis buffer is to decide the pH that will be most optimal for your protein and that is compatible with the first step in the protein purification process. Frequently used buffering components are for example Tris, phosphate and HEPES, as they have a good buffering capacity around physiological pH conditions.

It’s important to keep in mind that the pH of Tris is quite temperature-dependent, so ideally you will prepare your buffer at the same temperature as the one that will be used during the purification to assure the pH will be correct at the working temperature. Furthermore, Tris contains a potentially reactive amine group, so it might not be compatible with all downstream applications (e.g. when you plan to label your protein using an amine-reactive dye). HEPES interferes with the Lowry assay and can form radicals under certain experimental conditions. Phosphate buffers are incompatible with the use of divalent cations such as Mg²⁺ and Ca^2+.

The additives you will add to the buffer also depend on the protein’s specific requirements and the compatibility with the first purification step. The table below gives an overview of commonly used buffer additives.

Upon cell lysis, various proteolytic enzymes can be released, which could decrease the overall yield of recombinant protein. Therefore, the lysis buffer is often supplemented with a cocktail of protease inhibitors. If your first step in the purification process is an immobilized metal affinity chromatography step (for example, Ni-NTA or Talon), it might be necessary to use an EDTA-free cocktail of protease inhibitors to prevent stripping of the divalent metal cations from the resin. To degrade nucleic acids, benzonase or DNase can be added to the lysis buffer as well.