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2. Double immunodiffusion

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(A) Antiserum

Figure 1 (A) Schematic representation of the principle of conventional immunoelectrophoresis. 1, Electrophoresis of the sample in agar or agarose gels; 2, double immunodiffusion of the separated proteins and the corresponding antiserum. (B) Application of the technique to the study of specificity of various antisera. In all wells, fetal pig serum was applied. In the channels, the following antisera were assayed: 1, anti-fetal pig serum, 2, anti-transferrin; 3, anti-albumin; 4, anti-a-fetoprotein; 5, anti-aranti-trypsin; 6, anti-aracid glycoprotein; 7, anti-fetuin. (With permission from Lampreave, F, Gonzalez-Ramon N, Martinez-Ayensa S, Hernandez MA, Lorenzo HK, Garcia-Gil A and Pineiro A (1994) Electrophoresis 15: 672-676.)

corresponding antiserum and the plate is maintained in a humid, sealed box, for a period of between 24 and 48 h. Under these conditions, the combination of the radial diffusion of the proteins from the circular or ovoid spots obtained after the electrophoretic run, with the uniform diffusion of the antibodies, occurring in a perpendicular direction to the channel, produces arcs of precipitation in the different elec-trophoretic zones.

The IE patterns can be directly visualized or photographed, in the wet gel, by dark-field illumination. In this method the oblique light from a circular source placed below the plate is directed through the gel and transmitted at an angle of about 25°. The immuno-precipitates are visible as white lines on the dark background. To preserve the plates and before staining the immunoprecipitates, it is necessary to wash the gel plates extensively so that all the unprecipi-tated materials are removed. The wash is accomplished by immersing the gel plates in a buffered saline solution (0.01 molL"1 phosphate, NaCl 0.15 mol L"1 buffer, pH 7.4) for 1-3 days, and with several changes of liquid. Alternatively, a quick procedure can be carried out by placing several paper towels on the gel plate and applying a moderate pressure for some minutes. In this way, the liquid and all the soluble material are removed from the gel and absorbed by the paper towels. Then, the plate is submerged in the saline solution until the gel almost recovers its original thickness. Repeating this drying/soaking cycle, it is possible to achieve effective and rapid washing of the plates. Finally, the plates are dried and the immunoprecipitates stained, commonly with Coomassie blue or Amido black. The sensitivity of the method, using these conditions, allows the detection of proteins with concentrations ranging from 3 to 20 |g mL_1. Specific staining methods have been introduced to facilitate the identification of single proteins. For example, Sudan black can be used to detect lipoproteins and periodic acid (Schiff reagent) for polysaccharides and glycoproteins. There are also specific staining procedures for some proteins, such as ceruloplasmin, hemopexin, etc.

IE has been used to analyse complex mixtures of proteins from tissue extracts; to detect impurities during the monitoring of protein purifications; to detect differential expression of protein during growth and differentiation; to study the expression of single proteins during pathological situations and to detect protein polymorphisms. Figure 1B shows an example of the application of the IE technique. In the plate, the specificity of different antisera against six proteins isolated from fetal pig sera (albumin, a-fetoprotein, a1-acid glycoprotein, a1-antitrypsin, fe-tuin and transferrin) is analysed by attaching them to a fetal pig serum by this technique. As reference, the same fetal pig serum is analysed against a polivalent anti-fetal pig antiserum.

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Solar Panel Basics

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