Electrophoresis staining

Only a small percentage ( 3%) of a protein sample is labelled via fluorescent dye, and this addition to each protein alters the molecular mass by approximately 580 Da. In proteins over 30 kDa, this will not significantly alter the position where they resolve by 2DE. However, in smaller proteins this will significantly adjust positioning compared to the unlabelled proteins, but post-electrophoresis staining with a conventional stain allows further analysis. To date, a DIGE analysis of parasite proteins has not been published. 

Figure 9-9 IF of a serum containing an IgM kappa paraprotein. Lane , serum electrophoresis stained for protein lane 2, anti-[gG, Fc piece-specific lane 3, anti-IgA, a-chain specific lane 4, anti-IgM, jii-chain specific lane 5, anti-K light chain lane 6, anti-X, light chain. (Courtesy Katherine Boyer, Protein Laboratory, Hospital of the University of Pennsylvania, Philadelphia, Pa.)...

Both pre- and post-electrophoresis staining can be done. However, post-staining usually is preferred, because it does not alter the separation. Anilinonaphthalene sulfonate can detect 20 pg of protein. Dansyl chloride reacts with amines, amino acids, proteins, and phenols. Exposure of protein for 1 to 2 minutes at 100 °C to dansyl chloride produces a fluorescence capable of detecting 8 to 10 ng. Similarly, exposing primary amine-containing compounds to fluor-escamine at room temperature and an alkaline pH permits the detection of 6 ng of myoglobin. A newly popular compound is 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF). It can detect 1 ng of protein, and it is linear from 1 to 500 ng. 

Electrophoresis-polarography Globulins Electrophoresis, staining and elution Globulins ... 

Fig. 2. Electroblots of papain digestion fragments, resolved by a 15% SDS electrophoresis. Stained gel low molecular mass standards (1), papain digestion (2). Immunoblot incubated with monoclonal antibody CI9 (3). Autoradiogram after incubation with Zn (4), or with nick translated P-DNA 1 hr exposure (5) and 16 hr (6).

Most sample components analyzed with electrophoretic techniques are invisible to the naked eye. Thus methods have been developed to visualize and quantify separated compounds. These techniques most commonly involve chemically fixing and then staining the compounds in the gel. Other detection techniques can sometimes yield more information, such as detection using antibodies to specific compounds, which gives positive identification of a sample component either by immunoelectrophoretic or blotting techniques, or enhanced detection by combining two different electrophoresis methods in two-dimensional electrophoretic techniques. 

As an alternative to radiation, a stain such as ethidium bromide is used to visualize DNA. The ethidium may be incorporated into the stmcture of DNA either before or after electrophoresis. The gel is then visualized under a fluorescent lamp. 

Figure 7-11. Normal and pathologic patterns of lactate dehydrogenase (LDH) isozymes in human serum. LDH isozymes of serum were separated by electrophoresis and visualized using the coupled reaction scheme shown on the left. (NBT, nitroblue tetrazolium PMS, phenazine methylsulfate). At right is shown the stained electropherogram. Pattern A is serum from a patient with a myocardial infarct B is normal serum and C is serum from a patient with liver disease. Arabic numerals denote specific LDH isozymes.

Fig ure 3. Starch gel electrophoresis of hemoglobins. Tris-EDTA-boric acid buffer, pH 9.0. O-Dianisidine stain. 

Figure 5. Citrate agar electrophoresis of hemoglobins. The plate is stained with O-dimethyb...

Often these methods are not sensitive enough for quantitation of different CK isoenzymes. Konttinen and Somer (45) used electrophoresis on agarose to obtain sharper bands and increased sensitivity. Staining with nitro-blue tetrazolium was found to... 

Middle panel Cell wall proteins were isolated, 10 pgm of each resolved by non-denaturing polyacrylamide gel electrophoresis and PGl and PG2 isoforms detected by activity staining. 

The purity of the AE fraction was investigated by SDS-PAGE using Pharmacia PhastSystem with 10 - 15% SDS-gradient gels. Electrophoresis and silver staining of the proteins were performed as described by the manuals from Pharmacia. For determination of pi lEF 3-9 PhastSystem gels were used. 

Marshall, T. and Williams, K. M. (1987). Electrophoresis of honey Characterization of trace proteins from a complex biological matrix by silver staining. Anal. Biochem. 167,301-303. 

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