Chromogenic biotin

Nitration of the tyrosine rings in the four binding pockets of avidin or streptavidin can be done to increase the steric hinderance within the biotin binding sites (Morag et al., 1996). This process yields chromogenic proteins that have reduced binding affinity for biotin, thus allowing elution of biotinylated molecules under mild conditions. 

NHS ester Chromogenic Hydrophilic PEG3 Biotin group... 

Figure 18.15 NHS-chromogenic-PEG3-biotin contains an amine-reactive NHS ester that can be used to label biomolecules through an amide linkage. The chromogenic bis-aryl hydrazone group within the spacer arm of the reagent allows the degree of biotinylation to be quantified by measuring its absorbance at 354 nm. The compound also contains a hydrophilic PEG spacer, which provides greater water solubility.

Figure 18.16 NHS-chromogenic-PEG3-biotin reacts with amine groups in proteins or other molecules to form amide bond derivatives.

In a fume hood, dissolve NHS-chromogenic-PEXij-biotin in DMF at a concentration of 12.33 mM (2mg/200pl DMF). With mixing, add a quantity of the reagent to the protein solution to provide the desired molar excess (i.e., 10- to -20 fold excess). 

Measure the absorbance of the biotinylated protein solution at 354 nm. Use the molar extinction coefficient for the chromogenic group (e = 29,000 M-1cm-1) to determine the concentration of biotin present. To determine the molar ratio of biotin-to-protein, divide the molar concentration of biotin by the molar concentration of protein present (which may be determined by using the Coomassie assay or the BCA assay methods). 

Make it visible the fluorophore label can be visualized directly using fluorescent microscopy. The biotin label (see Sect. 6.2.1) can be detected using streptavidin conjugated with an enzyme the latter must be visualized through an enzyme chromogenic system. Incubate sections with an appropriate enzyme substrate until optimal color develops (see Sect. 2.3). 

The complexities of protocols for fluorescent and chromogenic in situ hybridization necessarily entail careful attention to controls. In particular, the possibility of native enzyme activity or the presence of endogenous biotin in the experimental tissue should be considered, though this can be addressed by exposing control tissue to the detection system in the absence of probe. The relative merits of digoxigenin versus biotin, and some of the technical problems associated with each, have been previously discussed (Chevalier et al., 1997 Luo and jackson, 1999). 

A cocktail (1 1) of two monoclonal anticyclin Dl/bc 1-1 antibodies were used P2D11F11 (diluted 1 40) and 5D4 (diluted 1 100) these two antibodies can be obtained from Vecta Laboratories, Inc., Burlingame, CA, and Immunotech, Westbrook, ME, respectively. The avidin-biotin immunoperoxidase detection system employing DAB is used as the chromogen (Ventana Biotek). After counterstaining in dilute Mayer s hematoxylin, the sections are dehydrated and mounted in Permount. 

The sections are treated with H202 and then incubated in the primary antibody at a dilution of 1 50. This is followed by sequential incubation in the biotinylated antimouse antibody and streptavidin-biotin-labeled complex. DAB is used for 5 min as the chromogen, and the sections are lightly counterstained with hematoxylin. Positive controls involve the use of the tissue known to express the antigen under study. Negative controls involve the replacement of the primary antibody with the diluent alone or with a non-immune serum. 

Laborious. Toxic chromogens. Need to block endogenous biotin activity. 

A variety of techniques (Fig. 1), which use a variable combination of antibody steps, enzyme labels, and chromogens, are employed in immunohistochemistry. The most common immunohistochemical methods are the PAP (peroxidase-antiperoxidase), ABC (avidin-biotin) peroxidase or alkaline phosphatase, and the APAAP (alkaline phosphatase-anti-alkaline phosophatase) techniques however,... 

Figure 1. Conventional immunohistochemical detection methods. Florseradish peroxidase (HRP) and alkaline phosphatase are commonly employed as enzymes for visualization with chromogen. A The polymer -based method in which dextran polymer is commonly used. B Streptavidin/biotin reaction-based methods including the labeled streptavidin (LSAB) and streptavidin-biotin complex (sABC) methods.

Immunohistochemical staining can be direct or indirect. Direct immunohis-tochemical staining methods utilize only a primary antibody, which may be conjugated to horseradish peroxidase, biotin, alkaline phosphatase, or other chromogens. In the case of biotin-labeled primary antibodies, avidin or strep-tavidin linked to peroxidase binds to the biotin allowing detection of reactivity of the test antibody with the tissue. Indirect immunohistochemical staining methods utilize secondary, tertiary, or even quaternary antibodies, any of which may be linked either to biotin or enzyme (e.g., peroxidase). 

Figure 2. Coronin 1C in melanoma. A) Immunohistochemical staining of the indicated tissue samples for Coronin 1C. S m sections from melanoma tumors were stained with a new Coronin 1C specific mAb (Roadcap and Bear, unpublished reagent) for 30min at 37°C with a steam heat-induced epitope retrieval, using the streptavidin/biotin method with an alkaline phosphatase label and Permanent Red (Dako) as the chromogen and then stained with hematoxylin. B) qRT-PCR analysis of the indicated melanoma cell lines treated with the Erk inhibitor U0126 or vehicle control for 24 hours.Dotted line indicates lx threshold.

It should be noted that the relationship between the final signal output and concentration of the analyte (dose-response) may be one of direct or inverse proportionality, and is dependent on the specific assay format. In addition, a number of different reporter enzymes may be used (e.g., horseradish peroxidase, alkaline phosphatase, p-galactosidase), along with a number of different signaling systems (e.g., substrates that yield chromogenic or fluorescent or chemiluminescent products, activation of signaling enzymes, amplification by biotin-avidin system or polymerase chain reaction). 

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