Detection by staining

Staining of macromolecules with suitable dyes is no doubt the most popular method of detection in different types of gel electrophoresis. It can, however, be used also with other sorbents of definite structure like paper, cellulose acetate, etc. Because the efficiency of the method is based on non-specific sorption differences, the choice of the dye must be such that it will be strongly bound to the separated macromolecule 

It has been mentioned already that destaining is positively the most time-consuming operation. Frequent exchanging of the destaining solution is necessary to ensure obtaining the results within a reasonable period of time. In a most primitive way electrophoretic destaining can be materialized in a beaker filled with destaining solution into which both electrodes and the gel to be destained are placed. The efficiency of this system is, however, low in comparison with the more sophisticated devices either commercially available or described in the literature [215-222]. 

We now present some widely used staining and destaining solutions. 

1% Amido Black lOB in 7% acetic acid solution Destaining and storage solution 7% acetic acid 

Staining is performed in test tubes (for rods) or troughs (for slabs) within 2-10 h. 

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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.)...

The band number refers to the position of migration on SDS-PAGE (see Figure 52-3). The glycophorins are detected by staining with the periodic acid-Schiff reagent. A number of other components (eg, 42 and 4.9) are not listed. Native spectrin is... 

Analyze the NHS-palmitate for purity using TLC on silica plates. Develop using a solvent mixture of chloroforrmpetroleum diethyl ether (bp 40-60°C) of 8 2. Excess NHS and NHS-palmitate may be detected by staining with 10 percent hydroxylamine in 0.1 N NaOH, followed after 2 minutes by a 5 percent solution of FeCl3 in 1.2 N HC1 (creates red colored spots). 

Visual examination of crystals using a light microscope does not indicate whether the crystals consist of only the protein or the protein-DNA complex. Therefore, the crystals are washed free of any uncrystallized DNA and protein several times with a solution containing the precipitant and any additives, etc. at the concentration and pH used for growing crystals (mother liquor). Finally, the crystals are separated from the mother liquor by microcentrifugation, dissolved in a suitable buffer, and analysed biochemically. The protein content is determined by SDS polyacrylamide gel electrophoresis, the protein concentration by BIO-RAD assay, and amino acid composition by mass-spectroscopy. The DNA can be detected by staining the gel with ethidium bromide or methylene blue (Jordan et al., 1985), whereas... 

Tuberculous meningitis—clinical manifestation is completely different, but otherwise the differentiation is more feasible using biochemical rather than cytological parameters. Sporadically, the agent is detected by staining for acid-fast... 

Disc gel electrophoresis yields excellent resolution and is the method of choice for analysis of proteins and nucleic acid fragments. Protein or nucleic acid bands containing as little as 1 or 2 ju,g can be detected by staining the gels after electrophoresis. 

Fig. 2. Time-window for the neuroprotection afforded by MS against MCAo-induced brain damage. Brain infarct volume produced by transient (2 h) MCAo followed by 22 h of reperfusion in rats treated with MS (150 mg/kg) or vehicle (PBS, 1 ml/kg) i.p., 1 h or 15 min before the induction of ischemia, or upon reperfusion. Infarct volume was detected by staining consecutive 2-mm-thick coronal brain slices with TTC as described in the methods section. Data from vehicle-treated animals were pooled together, being the infarct volume values not significantly affected by the treatment schedule. Values are expressed as mean S.E.M. indicates P < 0.05 versus vehicle (one-way ANOVA followed by Dunnett s posttest n — 4-6 rats per experimental group).

The presence of mycoplasmal DNA in the cell cytoplasm or attached to the cell membrane may be detected by staining with the fluoro-chrome Hoechst 33258 (Appendix 3). This intercalating dye fluoresces under ultraviolet light and this forms the basis of a very rapid test for mycoplasmas. 

Pechanek et al. (1982) determined ionic polysaccharides by fl migration through polyacrylamide and agarose gels and on cellulose acetate membranes the polyanions were detected by staining. At the dimensions found in gel micropores, pairs of surfaces create an adsorption potential ( ) 3.5 times that created at the same distance from a single surface (Void and Void, 1983). 

Figure 7.10 Agarose gel electrophoresis patterns for Ce(iv)/EDTA-induced site-selective hydrolysis of double-stranded DNA by Ce(iv)/EDTA and pcPNA additives. Bands were detected by staining with GelStar. (a) Site-selective hydrolysis of linearized PBR322 using pcPNAs. Lane 1, control lane 2, Ce(iv)/...

The CK isozymes may be separated by electrophoresis and detected by stains for their enzyme activity. CK-MB immunoassays have also been developed for use in humans that occasionally cross-react with some animal species, although these must first be shown to have appropriate cross-reactivity for the species tested. The LD isozymes have been commonly and readily separated by electrophoresis and stained by their enzymatic activity. For both CK and LD isozymes, their absolute activity is inferred from their relative staining on electrophoretograms and determination of total plasma enzyme activity, typically using an automated chemistry analyzer. 

This chck chemistry-based approach to ABPP even works in live animals to determine specific protein expression levels. As an example, PS-N3 (366) was injected into living mice to investigate ECH-1 expression in the heart tissue. One hour after injection, the mice were sacrificed, and ECH-1 was successfully detected by staining the crude heart-homogenate via Cu(l)-catalyzed bioconjugation using a Rh-alkyne tag 367 (Scheme 74B) [233]. 

Protein phosphorylation was carried out by a standard method as described in (7) with the exception that (32p)ppi was used as phosphate donor instead of (y- P)ATP. Isolated thylakoids were phosphorylated by illumination (500 pE m"2 s l) in the presence of 0.4 mM (32p)ppi (300 000 cpm/nmol PPi). Phosphorylation was terminated by adding 10% cold TCA. Thylakoids were washed, solubilized in SDS at 70 C, and fractionated by SDS-PAGE using a 12-22.5% acrylamide gradient (8). The thylakoid proteins were detected by staining the gel with Coomassie brilliant blue and radioactivity was located by autoradiography. 

Analysis of protein levels Total soluble leaf proteins were fi actionated in SDS-polyacrylamide gels and transferred to nitrocellulose. Immunodetection was performed usin antibodies followed by horseradish peroxidase-conjugated goat anti-rabbit antiserum and the peroxidase activity detected by staining with chloro-l-naphthol. 

Glycoproteins containing sialic acid may be detected by staining with a cationic carbocyanine dye before and after digestion with neuraminidase a change in the colour of the stain from blue to red-purple indicates the presence of sialic acid. The n.m.r. spectra of iV-acetylneuraminic acid and its methyl ester in DMSO and in water have indicated that the acid exists predominantly in the /S-form in solution. The finding supports the assumption that 7V-acetyl-neuraminic acid, produced by the enzymic cleavage of its a-ketosides, leaves the catalytic site of Vibrio cholerae neuraminidase as the j3-anomer. 

The aldehyde-modified HSA was analyzed via SDS-PAGE, using 12.5% polyacrylamide gel, and detected by staining with Coomassie Brilliant Blue (CBB). 

The induction of streptozotocin diabetes was associated with increased accumulation of lipid rich plaque in the arch, thoracic and abdominal aorta, as detected by staining for fat with Sudan IV. Treatment with the AGE-inhibitor, LR-90 had a modest but significant effect on plaque accumulation, especially in the thoracic and abdominal segments (Figure 3). No significant effect was observed in the arch, consistent with the known haemodynamic dependence of arch lesions... 

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