Glycoproteins staining

The successful glycosylation of the polypeptides was monitored via SDS-PAGE with visualization via coomassie blue and periodate staining. In periodate staining, the carbohydrates were oxidized to aldehydes via treatment with periodic acid, followed by treatment with Gel-Code glycoprotein staining solution (Pierce, Rockford, IL). 

This green fluorescent Pro-Q Emerald 300 glycoprotein-staining method can he combined with SYPRO Ruby for total protein staining, with sensitivity up to... 

A similar staining method using Pierce s GelCode Glycoprotein staining kit is described below ... 

Transfer gel to 25 ml of GelCode glycoprotein-staining reagent and incubate for 15 min (if crystals form in the reagent, remove them by centrifugation do not use heat to dissolve crystals). 

Protein samples with and without enzyme treatment are applied for SDS-gel separation and glycoprotein staining as described above. 

Figure 52-3. Diagrammatic representation of the major proteins of the membrane of the human red blood cell separated by SDS-PAGE. The bands detected by staining with Coomassie blue are shown in the two left-hand channels, and the glycoproteins detected by staining with periodic acid-Schiff (PAS) reagent are shown in the right-hand channel. (Reproduced, with permission, from Beck WS, Tepper Ri Hemolytic anemias iii membrane disorders, in Hematology, 5th ed. Beck WS [editor]. The MiT Press, 1991.)...

This process seems to reflect gradual increases in the intensity and density of labelled receptor cell bodies and their axons, followed by regional bulbar staining as synaptogenesis proceeds. However, a more extensive range of lectins needs to be examined before the implications of species differences in the membrane glycoproteins can be satisfactorily interpreted (Salazar and Quinteiro, 1998). 

Attempts to study the entry of ES products into cells using markers of fluid phase endocytosis yielded unexpected results. When larvae browse resistant IEC-6 cells in the presence of extracellular fluorescent dextran, dextran enters the cytoplasm of a significant proportion of the cells in the mono-layer (Butcher et al., 2000). The parameters of dextran entry are most compatible with the conclusion that larvae wound the plasma membranes of IEC-6 cells that is, they create transient breaches in the membrane that allow impermeant markers to enter the cell (McNeil and Ito, 1989). Wounding is considered to be a common occurrence in intestinal epithelia (McNeil and Ito, 1989). Injured cells are able to heal their wounds by recruiting vesicles to seal the breach (Steinhardt et al., 1994). In an experimental system, healing allows the injured cell to retain cytoplasmic dextran. In epithelial cell cultures inoculated with T. spiralis larvae, the relationship between glycoprotein delivery and injury of plasma membranes is not clear, i.e. dextran-laden cells do not always stain with Tyv-specific antibodies and... 

Golgi, in early neuroanatomical studies (1898) staining neurones by silver impregnation, observed a reticular apparatus which was crescent shaped and appeared to be linked through canaliculi. The structure was also seen in secretory cells. Between 1949 and 1954, Baker reported the presence of similar systems in unfixed cells examined by phase contrast. The structures could be stained by osmium tetroxide and probably contained lipid. They also stained for glycoprotein and alkaline phosphatase. Baker s confirmation of the existence of the... 

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. 

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