Horseradish peroxidase streptavidin conjugate

If p-galactosidasc is used to conjugate with an SMCC-activated avidin or streptavidin, then there is no need to thiolate the enzyme, since it contains sulfhydryls in its native state (Sivakoff and Janes, 1988 Fujiwara et al., 1988). For conjugations using horseradish peroxidase, alkaline phosphatase, or glucose oxidase, however, thiolation is necessary to add the requisite sulfhydryls. 

Streptavidin-horseradish peroxidase (HP) conjugate (Dako) Store at +4°C. Working solution dilute 1 600 in IX TBS prior to use. 

C. Conjugation of horseradish peroxidase (POase) Io antibodies or streptavidin... 

Streptavidin-horseradish peroxidase conjugate reagent (Amersham). 

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... 

Perhaps the most common conjugates of avidin or streptavidin involve attaching enzyme molecules for use in ELISA systems. As in the case of antibody-enzyme conjugation schemes (Chapter 10), by far the most commonly used enzymes for this purpose are horseradish peroxidase and alkaline phosphatase. Other enzymes such as (3-galactosidase and glucose oxidase are used less often, especially with regard to assay tests for clinically important analytes (Chapter 16). 

The CARD approach uses horseradish peroxidase to catalyze the deposition of tyramide molecules that are conjugated to an antihapten immunoglobulin or Streptavidin, which in turn, during the first incubation step, binds to the digoxigenin- or biotin-labeled nucleic acid target. Thus, the number of target sites for the next reaction step is markedly increased. 

In glycoprotein detection systems the carbohydrate portions of proteins are oxidized with sodium metaperiodate to generate aldehydes that can react with hydrazides. A biotin hydrazide is used to attach biotin onto the oxidized carbohydrates and horseradish peroxidase-conjugated streptavidin is used for chemiluminescence-based detection (Glycoprotein Detection Module, Amersham Biosciences, Uppsala, Sweden). 

PAP, peroxidase-antiperoxidase ABC, avidin-biotin conjugate APAAP, alkaline phosphatase antialkaline phosphatase B-SA, biotin-streptavidin PCNA, proiiferating cell nuclear antigen EPOS, enhanced polymer one-step staining (Dako) CARD, catalyzed reporter deposition HRP, horseradish peroxidase. 

Chemiluminescent detection is also used, mainly to detect antigen capture on antibody arrays by sequential incubations with biotinylated secondary antibodies and Streptavidin-conjugated horseradish peroxidase (HRP) [61]. Phosphorylation of arrayed kinase substrates in the presence of radiolabeled adenosine triphosphate (ATP) has been detected by autoradiography [62]. 

We have developed a hybridization approach to monitor the intracellular uptake and distribution of ASOs in cell culture experiments [R. Berg et al., unpublished]. Cells treated with ASOs were hxed in paraformaldehyde and then biotin-labeled complementary oligonucleotides were added in hybridization buffer. Detection was with an avidin-horseradish peroxidase conjugate, which cleaves diaminobenzidine to produce a colored precipitate within the cells. An alternative detection approach uses fluorescein-labeled streptavidin, which is visualized by fluorescence and/or confocal microscopy [S. Fard et al., manuscript submitted]. We are currently adapting this protocol for use on tissue and tumor sections taken from mice treated sys-temically with ASOs. 

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