Chromatofocusing

Fractionation by chromatofocusing separates proteins on the basis of their isoelectric points. This technique basically involves percolating a buffer of one pH through an ion-exchange column that is pre-equilibrated at a different pH. Owing to the natural buffering capacity of the exchanger, a continuous pH gradient may be set up along the length of the column. In order to achieve maximum resolution, a linear pH gradient must be constructed. This necessitates the use of an eluent buffer and exchanger that exhibit even buffering capacity over a wide range of pH values. The range of the pH gradient achieved will obviously depend on the pH at which the ion exchanger is pre-equilibrated and the pH of the eluent buffer. The sample is applied, usually in the running buffer, whose pH is lower than that of the pre-equilibrated column. After sample application, the column is constantly percolated with a specially formulated buffer that establishes an increasing pH gradient down the length of the column.

Upon sample application, negatively charged proteins immediately adsorb to the anion exchanger, while positively charged proteins flow down the column. Owing to the increasing pH gradient formed, such positively charged proteins will eventually reach a point within the column where the column pH equals their own pI values (their isoelectric points, i.e. the pH value at which the protein has an overall net charge of zero). Immediately upon further migration down the column such proteins become negatively charged, as the surrounding pH values increase above their pI values; hence, they bind to the column. Overall, therefore, upon initial application of the elution buffer, all protein species will migrate down the column until they reach a point where the column pH is marginally above their isoelectric points. At this stage they bind to the anion exchanger. Proteins of differing isoelectric points are thus fractionated on the basis of this parameter of molecular distinction.

The pH gradient formed is not a static one. As more elution buffer is applied, the pH value at any given point along the column is continually increasing. Thus, any protein that binds to the column will be almost immediately desorbed, as once again it experiences a surrounding pH value above its pI and becomes positively charged. Any such desorbed protein flows down the column until it reaches a further point where the pH value is marginally above its pI value, and it again rebinds. This process is repeated until the protein emerges from the column at its isoelectric point. To achieve the best results, the isoelectric point (pI value) of the required protein should ideally be in the middle of the pH gradient generated. Chromatofocusing can result in a high degree of protein resolution, with protein bands being eluted as tight peaks. This technique is particularly effective when used in conjunction with other chromatographic methods during protein purification. Most documented applications of this method still pertain to laboratory-scale procedures. Scaling up to industrial level is somewhat discouraged by economic factors, most notably the cost of the eluent required.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

Get My Free Ebook


Post a comment