P-galactosidase conjugate

Add 100 pL/well of appropriate second-layer reagent diluted in TMT Dilute the stock HRP-anti-Ig conjugates (from the manufacturer) 1 in 200, stock protein A-HRP solution (1 mg/mL) 1 in 200, and stock P-galactosidase conjugates 1 in 100 (see Note 3). 

Add 100 pL of p-galactosidase conjugated secondary antibody diluted in PBS/ FCS. Incubate at room temperature for lh. 

Streptavidin-P-galactosidase conjugate. We routinely use the product from Boehringer Mannheim (Mannheim, Germany). It is supplied as a powder which is reconstituted with water and then, to avoid repeated freeze-thawing, this is stored in aliquots at -20°C. These are stable for many months and, once thawed, each aliquot is stable at 4°C for up to about 4 wk. Before use, aliquots of this stock solution are diluted xl0,000 into PBSTw containing 1% w/v BSA. 

A Witkowski, et al. Preparation of P-Galactosidase conjugates for competitive binding assays by posttranslational modification of recombinant proteins. Anal Chem 67 1301, 1995. 

Biotinylated goat anti-rabbit IgG (2° antibody diluted 1 200 in PBS/0.2%BSA Vector Laboratories, PK6101) for 60 min at room temperature. Streptavidin-P-galactosidase conjugate (diluted 1 200 in PBS/0.2% BSA Roche 1112481). 

Wash the sections in PBS containing 0.2% BSA (3 x lOmin), then incubate with streptavidin-P-galactosidase conjugate for 60min (1 200 dilution). 

The following protocol illustrates the use of SIAB in preparing antibody-enzyme conjugates using P-galactosidase. 

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

If the molecule being conjugated to avidin is P-galactosidase or other free thiol-contaming oligonucleotide or protein, NEM treatment is not performed. 

Secondary antibody conjugated to p-galactosidase for example, a p-galactosi-dase conjugated anti-mouse antibody (Southern Biotechnology dilution 1 400). 

The ProLabel peptide can be chemically conjugated or recombinantly fused to various biomolecules. To probe molecular interactions, EEC assays are based on a competition between the free and the ProLabel peptide-conjugated form of the biomolecule involved in the interaction under study. Bound to its interaction partner, the ProLabel-labelled biomolecule is not able to complement with the Enzyme Acceptor. Therefore the signal generated by the P-galactosidase is proportional to the concentration of the free biomolecule in the assay."... 

In most of the assays described the detection of rat or mouse MAb depends on the use of a second antibody reagent specific for F (ab )2 or heavy chain isotype. These second antibodies are detected because they have either a radiolabel ( I), fluorescent tag (fluorescein), or are conjugated to an enzyme (e.g., alkaline phosphatase, peroxidase, or p-galactosidase) either direcdy or indirecdy via a biotin-streptavidin bridge. As examples of these procedures, two altemadve types of methodology, i.e., radioimmunoassay... 

Enzyme conjugated second antibody We routinely use affinity purified F(ab )2 species specific antibody conjugated to P-galactosidase (from Amersham, Slough, UK). This is supplied as a solution that should typically be diluted 1 in 500 into PBSTw containing 1% (w/v) BSA, 1 mM i-mercaptoethanol, and 10 mMMgClj. 

Given this information, researchers have devised donor and acceptor components of the p-galactosidase subunit. The enzyme donor is conjugated to antigen, and becomes the indirect label in a competitive immunoassay. If this labeled antigen is not bound to antibody, then the combination of donor and acceptor result in a complete subunit that can combine with three other complete subunits to form an active tetramer. If the labeled antigen is antibody-bound, steric effects prevent donor-acceptor combination, so that the generation of active enzyme is not possible from this potential subunit. The concepts in this assay are shown in Eq. 6.18 ... 

In solid-phase EIA, the influence the solid phase has on the enzyme (Section 9.2.2) should be minimal. Conjugation should be easy and the conjugates should be active and stable. These are undoubtedly major factors for the frequency of the selection of horseradish peroxidase (POase) or P-galactosidase (BGase). Alkaline phosphatase (AP-ase), which is difficult to conjugate in defined form (extensive polym-... 

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