Liquid Chromatography Resolves Proteins by Mass Charge or Binding Affinity

A third common technique for separating mixtures of proteins, as well as other molecules, is based on the principle that molecules dissolved in a solution will interact (bind and dissociate) with a solid surface. If the solution is allowed to flow across the surface, then molecules that interact frequently with the surface will spend more time bound to the surface and thus move more slowly than molecules that interact infrequently with the surface. In this technique, called liquid chromatography, the sample is placed on top of a tightly packed column of spherical beads held within a glass cylinder. The nature of these beads determines whether the separation of proteins depends on differences in mass, charge, or binding affinity.

Gel Filtration Chromatography Proteins that differ in mass can be separated on a column composed of porous beads made from polyacrylamide, dextran (a bacterial polysaccha-ride), or agarose (a seaweed derivative), a technique called gel filtration chromatography. Although proteins flow around the spherical beads in gel filtration chromatography, they spend some time within the large depressions that cover a bead's surface. Because smaller proteins can penetrate into these depres sions more easily than can larger proteins, they travel through a gel filtration column more slowly than do larger proteins (Figure 3-34a). (In contrast, proteins migrate through the pores in an electrophoretic gel; thus smaller proteins move faster than larger ones.) The total volume of liquid required to elute a protein from a gel filtration column depends on its mass: the smaller the mass, the greater the elution volume. By use of proteins of known mass, the elution volume can be used to estimate the mass of a protein in a mixture.

Ion-Exchange Chromatography In a second type of liquid chromatography, called ion-exchange chromatography, proteins are separated on the basis of differences in their charges. This technique makes use of specially modified beads whose surfaces are covered by amino groups or car-boxyl groups and thus carry either a positive charge (NH3+) or a negative charge (COO-) at neutral pH.

The proteins in a mixture carry various net charges at any given pH. When a solution of a protein mixture flows through a column of positively charged beads, only proteins with a net negative charge (acidic proteins) adhere to the beads; neutral and positively charged (basic) proteins flow unimpeded through the column (Figure 3-34b). The acidic proteins are then eluted selectively by passing a gradient of increasing concentrations of salt through the column. At low

(a) Gel filtration chromatography

(c) Antibody-affinity chromatography

Layer sample on column

■ Large protein

■ Small protein

■ Large protein

■ Small protein

Polymer gel bead

Collect fractions

10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook


    Do small or large proteins travel faster through gel filtration columns Why?
    7 years ago

Post a comment