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Densitometry

After electrophoresis, a stained gel is passed through the optical system of a densitometer to create an electrophoregram, a visual diagram or graph of the separated bands. A densitometer is a special spectrophotometer that measures light transmitted through a solid sample such as a cleared or transparent but stained gel. Using the optical density measurements, the densitometer represents the bands as peaks. These peaks compose the graph or electrophoregram and are printed on a recorder chart or computer display. Absorbance and/or fluorescence can be measured with densitometry.

An integrator or microprocessor evaluates the area under each peak and reports each as a percent of the total sample. If the electrophoresis is for separation of serum proteins, the concentration of each band is derived from this percent and the total protein concentration. If the electrophoresis is for separation of enzymes, the enzyme activity of each band is derived from this percent and the total enzyme activity.

The densitometer scan below depicts the separated bands from a serum sample electrophoresis. The SPIFE 3000, Helena Laboratories, electrophoresis splits the beta zone into two fractions for easier detection of small beta-migrating monoclonal gammopathies. The densitometer scan from this electrophoresis shows five bands with two peaks in the beta band. Recall the order of protein fractions from left to right is: Albumin, alpha 1, alpha 2, beta, and gamma.

Densitometer Scan of Serum Protein Electrophoresis