Staining carbohydrates

Periodate followed by Schiff reagent has been successfully used for staining carbohydrate structures on histological slides,188 189 but the possibility of interference by amino acids has been pointed out.190... 

Solubilization of erythrocyte ghosts in 1 % sodium dodecyl sulfate, followed by SDS-polyacrylamine gel electrophoresis and staining with Coomassie blue reveals more than 10 well defined protein bands. When staining is performed with periodic acid-Schiff reagent (PAS), which stains carbohydrates, four bands (PAS bands) are revealed. 

This is an extracellular deposition of an insoluble protein, which has adopted a (3-sheet structure due to an unknown event that induced misfolding of unstable proteins. The name amyloid has been given according to the amyloid staining properties, which are similar to carbohydrate deposits, e.g., amyloid can be identified with Congo red and seen under polarized light (birefringence test). 

Based on its nature (aqueous solutions, physiological conditions, well-investigated labeling, and staining reactions) and the historical transition from slab-gel electrophoresis to CE, the main targets are biological and bioequivalent samples such as proteins, peptides, polynucleotides, oligonucleotides, and carbohydrates. 

Figure 3. Gel electrophoresis of cotton dust fraction 4 stained for protein (left) and carbohydrate (right). Conditions described in Material and Methods.

A number of histologic and histochemical changes in current-year needles of ponderosa pine were detected after five to seven daily exposures to ozone at 0.45 ppm for 12 h each day. Chloroplasts and carbohydrate stain accumulated in the peripheral portions of mesophyll cells concurrently, the homogeneous distribution of proteins and nucleic acids was disrupted, and add phosphatase activity increased. Cell wall destruction occurred in mesophyll cells after appredable intracellular damage. 

Glycoprotein Carbohydrate Analysis. Gels from PAGE were fixed with iso-propanol-acetic acid-water (25 10 65 v v v), stained with 0.2% thymol in the fixing solution, and washed two or three times with the same fixing solution. This was followed by staining with concentrated H2S04-ethanol (4 1 v/v) at 35°C for 2-3 h (72). 

This procedure is not solely specific for carbohydrate side chains of proteins. Unglycosylated proteins may also be stained. To identify glycosylated proteins, the sample should be run in at least two identical lanes cut the gel and stain a lane with the common protein silver stain (Protocols 2.4.2.1 to 2.4.2.4) and the other lane by the described method. Compare pattern and intensity to identify glycoproteins. Glycosylated macromolecules are also stainable with Schiff s reagent (Protocol 2.4.4.1), but with less sensitivity. 

Enzymes. The most substantial advance in textile cleansing since the introduction of synthetic detergents has occurred through the introduction of low levels of enzymes into laundry detergent and presoak products. Both proteolytic and amylolylic enzymes are used by the industry to hydro yz.e protein and starch so that the smaller soil fragments are easier to remove. They are effective on stains with protein and carbohydrate substituents (such as body soils, many food stains, grass stains, blood, and many others). Enzymes are catalytic and specific and thus must be used at low levels in... 

The first and fastest tests simply categorize product components as protein, lipid, carbohydrate, ions or nucleic acids. Many of these techniques have been documented elsewhere for biological materials [18,20,22,23,29,30] but are repeated here to emphasize their utility in all staining, especially in the more difficult foods such as process cheese, lunchmeats etc., which are heavily buffered. Where such is the case, the pH of the unembedded product must be matched to that of the dye so as not to confound interpretation. 

Protein concentrations were determined according to the method of Lowry et al. (30). Electrophoresis of proteins in polyacrylamide gels was carried out at 4°C, using the discontinuous buffer system No. 1 described by Maurer (31) and modified by Emert et al. (1). Protein was stained with 0.1% Coomassie Brilliant Blue R250 in a water-acetic acid-methanol (45 10 45) solution. Carbohydrates were stained with the periodic acid-Schiff (PAS) reagent using the method described by Lang (32). 

Figure 11. Polyacrylamide disc gel electrophoretic patterns of extracellular proteins produced by T. reesei QM 9414. The sample applied to the gel on the left was 130 fig extracellular protein from T. reesei my-celia grown on 1% Avicel (29), that applied to the gel on the right was 120 fig extracellular protein produced from sophorose-incubated mycelia. The bands shown here were stained for protein with Coomassie Blue and could, in all cases, also be stained for carbohydrate with the periodic acid-Schiff reagent.

Fig. 2. Treatment of cell cultures with deoxymannojirimycin (DMM) inhibited processing of the complex carbohydrate moiety of the epsi-APase but did not affect excretion into the medium. Three-day-old cells grown —Pi as previously described (Goldstein etal., 1988a) were treated with 0.1 mM DMM for 24 h which resulted in inhibition of processing of the complex carbohydrate moiety of the epsi-APase. Culture-medium proteins from +DMM or -DMM treatments were precipitated with 50% acetone, separated via SDS-PAGE and activity stained (also as previously described, Goldstein etal., 1988b). As shown here, inhibition of carbohydrate processing caused a visible increase in apparent molecular mass but did not inhibit the excretion of epsi-APase into the medium.

Weisel, J. W. (1986a). The electron microscope band pattern of human fibrin Various stains, lateral order, and carbohydrate localization. /. Utirastruct. Mol. Struct. Res. 96, 176-188. 

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