Immunoglobulin biotinylated

Anti-anaphylactic activity. Water extract of the dried fruit, at a concentration of 1 pg/ mL, produced weak activity on the rat Leuk-RBL 2H3 vs biotinyl immunoglobulin E-avidin complex-induced degranulation of (3-hexosaminidase 5 Anti-androgenic effect. Ethanol (95%) extract of the aerial parts, administered intraperitoneally to castrated mice at a dose of 2 mg/animal, produced strong activity The dried leaf, smoked by 13 male adults for 21 days, was inactive . 

With mixing, add a quantity of the sulfo-NHS-biotin solution to the protein solution to obtain a 12- to 20-fold molar excess of biotinylation reagent over the quantity of protein present. For instance, for an immunoglobulin (MW 150,000) at a concentration of 10 mg/ml, 20 pi of a sulfo-NHS-biotin solution (containing 8 X 10-4 mmol) should be added per ml of antibody solution to obtain a 12-fold molar excess. For more dilute protein solutions (i.e., 1-2 mg/ml), increased amounts of biotinylation reagent may be required (i.e., 20-fold molar excess or more) to obtain similar incorporation yields as when using more concentrated protein solutions. 

Add 0.3 mg of sulfo-NHS-SS-biotin (Thermo Fisher) to each ml of the antibody solution. To measure out small amounts of the biotinylation reagent, it may be first dissolved in water at a concentration of at least 1 mg/ml. Immediately transfer the appropriate amount to the antibody solution. This level of sulfo-NHS-SS-biotin addition represents about an 8-fold molar excess over the amount of antibody present. This should result in a molar incorporation of approximately 2-4 biotins per immunoglobulin molecule. 

Another popular tag for use with immunoglobulins is biotin. Modification reagents that can add a functional biotin group to proteins, nucleic acids, and other molecules now come in many shapes and reactivities (Chapter 11). Depending on the reactive group present on the biotinylation... 

A very interesting approach was presented recently by Niemeyer et al. [113]. They prepared covalent DNA-streptavidin conjugates to which biotinylated alkaline phosphatase, beta-galactosidase, and horseradish peroxidase, as well as biotinylated anti-mouse and anti-rabbit immunoglobulins, were coupled. Immobilization of DNA-streptavidin conjugates was performed by hybridization with the complementary oligonucleotides, bound to the surface. It was demonstrated... 

The above represent the past and present of the most common enzyme-mediated methods of antigen detection. There are alternate procedures available, involving such methods as antibiotin antibody steps that combine the avidin-biotin systems with a further antibiotin/antienzyme sandwich for still greater sensitivity. Also, there are methods that follow a PAP procedure with a biotinylated antibody to the PAP immunoglobulin followed by ABC detection (15). The obvious problem created with this approach is the tremendous... 

Fig. 1. Diagram illustrating the molecular interactions of the ABC procedure. The primary antibody against the antigen of interest is linked to the avidin-biotinylated peroxidase complex via a biotinylated secondary antibody raised against immunoglobulin of the animal species used to generated the primary antibody (A, immunoglobulin , biotin A, avidin , peroxidase).

Many biotinylated secondary antibodies from various species that are reactive against various species immunoglobulin can now be pooled. Some manufacturers provide pooled or universal secondary reagents, which can be used regardless... 

Incubate grids for 30 min at room temperature in colloidal gold conjugated with protein A, protein G, immunoglobulin, or if a biotinylated secondary antibody was used, streptavidin (see Note 6). 

Even greater flexibility was achieved by genetic fusion of streptavidin with protein A [153,154]. Protein A specifically binds the Fc domain of IgG immunoglobulins of almost all mammals without inhibiting the antigen binding activity of the antibody. The streptavidin-protein A fusion construct was used for the assembly of complexes of biotinylated P-galactosidase and different monoclonal antibodies specific for tumour cell receptors. As a result these complexes were efficiently delivered into several cancer cell lines [154]. 

The following protocol is an example for the biotinylation of immunoglobulins in a molar ratio of 1 mole IgG to 5 moles biotin. Depending on the kind of protein, ratios up to 1 20 are possible. 

Biotinylated proteins such as immunoglobulins and serum albumins or biotinylated oligonucleotides can bind through affinity interactions streptavidin-functiona-lized Au NPs [156, 157]. 

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