CLINICAL PATHOLOGY
Serum Protein
Electrophoresis
Dr Emma Hooijberg BVSc GPCert ( SAP ) DipECVCP Department of Companion Animal Clinical Studies , University of Pretoria emma . hooijberg @ up . ac . za
How does this method work ?
Serum proteins come in different shapes and sizes , and also have different charges . Albumin , for example , is the most negatively charged protein , while immunoglobulins have a slight positive charge . Serum protein electrophoresis ( SPE ) separates proteins based on their size and charge . The most common method uses agarose gel as a platform . The gel provides a 3-D matrix with large pores ( compared to the size of proteins ). A serum sample is placed into the gel , and an electrical current is applied across it . The more negatively charged proteins migrate through the gel to the cathode ( positive charge ) and the more positively charged proteins , to the anode ( negative charge ).
This results in a separation of all the proteins into bands . A stain is applied to the gel , making the bands visible . The gel is then placed into a scanner , which converts the density of each band into curves , which appear as the typical fractions of the electrophoretogram ( Figure 1 ). / Each curve / fraction represents a percentage of all protein present in serum . This percentage is calculated by a computer programme , and is multiplied by the total protein concentration to give the concentration of each fraction . The fractions , apart from albumin , have traditionally been allocated a Greek letter and a number – alpha-1 , alpha-2 , beta-1 , beta-2 and gamma . It has been determined ( through proteomic studies using other methods ), which major proteins are found in each fraction , as illustrated below in Figure 1 .
Figure 1 : SPE tracing showing the different fractions . Specific proteins migrate to certain fractions based on their charge and size
SPE can be used to give an indication of which groups of proteins are increased or decreased , but does not generally help to identify changes in specific proteins . It should be noted that SPE is not a direct measurement of the concentrations of different proteins , although it is a fairly accurate estimate . It is also important to remember that each fraction contains many different proteins of similar but not identical charge and size – this is why the fractions have broad bases .
When should it be used ? SPE is indicated in the following scenarios : 1,2
1 . Determination of albumin in some exotic species : the assay ( bromocresol green reaction ) used to vet360
Issue 03 | JUNE 2017 | 32