Protein-specific staining

Because CE uses online optical detection, artifacts can result in the form of system peaks. These often originate from the sample or the interfaces between the sample and the separation buffer because any species that absorbs at the detection wavelength wfll generate a response. This differs from protein slab gel electrophoresis where detection specificity is governed by a protein specific stain. It is not uncommon, for example, for buffer species present in the sample but not in the separation buffer to generate system peaks. However, clinical serum protein electrophoresis provides one example where artifacts are elimmated by CE. 

Figure 14 6 Silver-stained SDS-PAGE gel of PatA binding proteins. Lane 1, sample 1 nonspecific proteins captured by the streptavidin-agarose resin Lane 2, sample 2 proteins affinity captured by the presence of B-Pat A Lane 3, sample 3 affinity capture of target proteins was blocked by prior addition of free PatA before incubation with B-PatA. The two arrows point to two proteins specifically detected in sample 2 versus sample 1, which were also lost due to competition in sample 3, with apparent molecular weights of 38 and 48 kDa.

Smithies vertical starch gel electrophoresis (S7) separates the plasma proteins more distinctly than any other method. If the Hp concentration is normal, the Hp type can generally be recognized directly after the staining for proteins, but sensitive and more specific staining for heme groups, e.g., benzidine, o-dianisidine (04), and malachite green (N5) are preferable. This technique consumes more hydrolyzed starch than the simpler original horizontal electrophoresis technique (S5). 

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. 

The first step in immunochemical detection of proteins after electrotransfer is blocking the support with an inert material to inactivate further non-specific binding of protein. The blocking reagent should cover the membranes at those areas where no blotted protein is bound and should not react with any of the reactants of immunochemical detection cascade as indicated by no non-specific staining, i.e., resulting in blank background of the membrane. 

Specificity Stains should be protein specific and be able to differentiate different classes... 

It is now recognised that a substantial number of proteins, especially enzymes, are polymorphic in that they exist in the cell as multiple molecular forms differing in certain of their physico-chemical properties. Each form of a polymorphic enzyme is called an isoenzyme or isozyme. Electrophoretic techniques provide convenient methods whereby this protein heterogeneity can be investigated and the approach has been widely exploited to characterise parasites. In short, aqueous parasite extracts are electro-phoresed, or focused isoelectrically, and separated proteins are stained generally (usually with Coomassie Blue) or more specifically with a histochemical (enzyme) stain (the zymogram technique). Further details of individual procedures and the use of the approach in parasite identification are to be found in a number of recent reviews (104,258,413,536,615,856). 

Focal cytoplasmic staining is seen, particularly in intermediate and superficial layers of the epidermis. May be caused by passive absorption of plasma proteins into degenerating epidermal cells. This observation is rare and should not interfere with interpretation of specific staining. 115-121... 

Figure 7.5 Electrophoretic patterns of erythrocyte lysates bearing abnormal hemoglobins. C, A2, S, F, Al7 Barts, and H are hemoglobins C, A2, S (sickle), Aa (normal), Barts and H, respectively. Electrophoresis was done at pH 8.6, the medium was stained with a protein-specific dye, and the bound dye was quantitated densitometrically. Absorbance is on the y axis, distance moved, on the x axis, Shaded areas represent abnormal patterns. (From a circular by Gelman Instrument Co., Ann Arbor, MI, by permission of the copyright holder.)...

Similarly, RNA molecules can be separated by gel electrophoresis, and specific sequences can be identified by hybridization subsequent to their transfer to nitrocellulose. This analogous technique for the analysis of RNA has been whimsically termed Northern blotting. A further play on words accounts for the term Western blotting, which refers to a technique for detecting a particular protein by staining with specific antibody (Section 4.3.4). Southern, Northern, and Western blots are also known respectively as DNA, RNA, and protein blots. 

There are many specific methods available that are suitable for a certain set of conditions or for a particular experimental aim (Patterson, 1979 Griffiths, 1985). These include radiochromium labelling (radiochromium binds to viable protein and is released on cell damage/death), cellular ATP, intracellular volume (by ratios of radiochemicals), specific stains (e.g. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), triphenyltetrazolium chloride, neutral red), redox potential, turbidity, calorimetry (Griffiths, 1985) and biomass monitors. 

The high detectability of these techniques may be due to the presence of multiple biotin molecules on the proteins. Non-specific staining can be considerable at high concentrations of ABC which may be related to the fact that biotin is an important coenzyme for transcarbamylation and may be present in some tissues to bind ABC. Pretreatment with avidin and biotin, respectively, prior to the serological detection, could remedy this problem in some cases. 

Kinases FP, TR-FRET, IMAP, thermal melt Lysed—FRET, AlphaScreen , Alpha-LISA Intact— PathHunter kinase [47] Specific a-P-protein mAb staining fixed and permeabilized cells... 

Protein-protein or protein-DNA/RNA EP, ERET, TR-FRET, BRET, ECL, AlphaScreen BRET, reporter gene BRET, localization, specific a-protein mAb staining on fixed-permeabilized cells... 

The location of individual proteins or protein subunits has been established by analyzing pure proteins and immunoprecipitates prepared using specific antisera, or by co-electrophoresis. Proteins containing cysteine residues can be specifically labeled using [ I]iodoacetamide and can be located autoradiographically. If analysis is performed under nondenaturing conditions, specific stains are available to locate certain types of protein, e.g., glycoproteins (A14, H12) and enzymes. 

---