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Spife Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Spife and links to relevant pages within the course.

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Automated systems for protein electrophoresis are available for large volumes of samples for electrophoresis. An automated system is capable of separating 10-100 samples simultaneously. There are several different automated systems and the number of process steps that are automated varies. Automated steps may include reagent addition, sample application, electrophoresis separation, staining, and detection.

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Immunofixation Electrophoresis

An agarose gel electrophoresis first separates the proteins in a serum sample. Antiserum against the protein of interest is spread directly on the gel. The protein of interest precipitates in the gel matrix. After a wash step to remove other proteins, the precipitated protein is stained. This method is qualitative and is used to identify proteins found in multiple myeloma.Below is the immunofixation electrophoresis gel from a serum sample analyzed on SPIFE 3000, Helena Laboratories. After electrophoresis, the precipitated proteins are stained with Acid Violet, a stain developed and used by Helena Laboratories. The SP lane represents a routine serum protein electrophoresis of this specimen. On the next three protein separations, antiserum against IgG, IgA, and IgM were applied to the G, A, M lanes respectively. Antiserum to kappa light chain was added to the next protein separation and antiserum to lambda light chain to the last protein separation.

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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.

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