1. Buffers for Biochemical ReactionsBY SREEREMYA.S

2. Buffers often are overlooked and taken forgranted by laboratory scientists until the daycomes when a bizarre artifact is observed and itsorigin is traced to a bad buffer. Although mistakes in the composition of buffershave led occasionally discoveries such as thecorrect number of human chromosomes(Arduengo, 2010), using the proper buffer,correctly prepared, can be key to success in thelaboratory 3. Exclusion by biological membranes. This isnot important for all biochemical reactions.However, if this is an important criterion for yourparticular experiment, it is helpful to rememberthat zwitterionic buffers (positive and negativecharges on different atoms within the molecule)do not pass through biological membranes.Examples of zwitterionic buffers include MOPSand HEPES; Tris and phosphate buffers do notisomerize into zwitterions. 4. Minimal salt effects. In other words, the buffercomponents should not interact or affect ionsinvolved in the biochemical reactions beingexplored. 5. Minimal effects on the dissociation from changesin temperature and concentration. Usually there issome change in the dissociation with a change inconcentration. If this change is small, stock solutionsusually can be diluted without changing the pH. However, with some buffers, changes in concentrationhave more effect on dissociation, and stock solutionscannot be diluted without significantly affecting pH. For instance, the pH of Tris decreases approximately0.1 pH unit per tenfold dilution, and the pH couldchange dramatically if you dilute a working solutionand are at the limits of the optimal buffering range ofthe Tris (optimal buffering range pH 7.39.3 at 20C).Note that Tris is not one of Goods buffers. 6. A pKa between 6 and 8. Most biochemicalexperiments have an optimal pH in the range of68. The optimal buffering range for a buffer isthe dissociation constant of the weak acidcomponent of the buffer (pKa) plus or minus pHunit. Solubility in water. Biological reactions, forthe most part, occur in aqueous environments,and the buffershould be water-soluble for this reason. 7. Prepare Buffers at the Appropriate Temperature andConcentration Because changes in temperature can beassociated with a shift in dissociation, prepareyour buffers at the temperature at which you willbe performing your experiments. If yourexperiment involves a change in temperature,choose a buffer with a pKa that accommodates it. Changes in concentration also can be associatedwith a shift in dissociation, so if you plan tomaintain buffer stock solutions, make sure thatthe pH adjustment is made after you have dilutedthe stock to the desired concentration andequilibrated it at the appropriate temperature. Or,at the very least, check the pH after dilution.