Buffers

Buffers are solutions that resist changes in pH when acidic or basic components are added. It neutralizes a small amount of added acid or base to keep the pH of the solution relatively stable. This is important for processes and reactions that require a certain stable pH range. The buffer solution has a working pH range and capacity that determines how much acid / base can be neutralized before the pH changes and the amount it changes.

Thumbnail: acidic solution of weak acid (pKa = 7) was titrated with base. License image (public domain; Lasse Havelund)

Radioimmunoprecipitation assay buffer (RIPA buffer) is a lysis buffer to lyse cells and tissues for radioimmunoprecipitation assay (RIPA). Since this buffer contains ionic detergent SDS and sodium deoxycholate as active ingredient, it is more modified than NP - 40 or Triton X - 100 and destroys the nuclear membrane in the preparation of nuclear extract . The RIPA buffer has a low background, but it may denature the kinase

The name of RIPA buffer comes from radioimprecipitation, which is the original application that it developed. This isotope analysis method is rarely performed in today's laboratory, but the acronym for this lysis buffer formulation is often used. Since the RIPA cell lysis reagent contains three nonionic and ionic surfactants, it is very effective for extracting proteins from various cell types. A disadvantage of this detergent formulation compared to other lytic reagents is that it is relatively incompatible with certain downstream applications.

Many components can be used as biological buffering agents. The most commonly used buffer components have near neutral pKa as they can be used at physiological pH. Table 1 lists the four most common biological buffers, the pH range in which they can be used, and the advantages and disadvantages that may affect use in protein purification. Typically, these buffers are used at concentrations above 25 mM to ensure adequate buffering capacity. Protease inhibitors are typically added to the lysis buffer and at an early stage of the purification protocol to prevent endogenous proteases from degrading the target protein. Since most or all of the contaminating proteases have been isolated from the target protein, it is generally not necessary to perform these operations at later stages of purification. Metal chelators such as EDTA and EGTA are usually added to storage buffer