Conjugates antidigoxigenin/alkaline phosphatase

Unfortunately, there is no quantitative method to measure digoxigenm-incorporation into the final probe, short of spotting the probe onto a membrane and testing with antidigoxigenin/alkaline phosphatase conjugate. 

Antidigoxigenin solution This solution consists of 50 mL of blocking buffer with 2.5 pL of antidigoxigenin/alkaline phosphatase conjugate (Boehringer Mannheim [Mannheim, Germany] 1093 274). This solution should be made up immediately before use. 

The visualization of hybridization between the RNA probe and the DNA fragments on the blot is based on an enzyme-linked immunoassay. An antidigoxigenin/alkaline phosphatase conjugate is bound to the digoxigenin component of the probe and then incubated with the substrates X-phosphate and nitroblue tetrazolium (see Section 2.) under alkaline conditions, which will result in purple-blue precipitates on the membrane within a couple of hours. Color detection thus saves time and money as it does not require X-ray films, cassettes, and intensifier screens. For a permanent record the result can be photocopied or photographed. 

ABC (antibody-conjugate) solution Dilute antidigoxigenin/alkaline phosphatase conjugate (polyclonal sheep antidigoxigenin Faj,-fragments) to 150 mU/mL in 100 inL TN buffer. Prepare on the day of use. This dilution is stable for 12 h (at 4 C). Bring to room temperature before use. 

Put on the section a drop of antidigoxigenin antisera conjugated to alkaline phosphatase, diluted 1/500 with buffer 1. Incubate 2 h at room temperature see Notes 8 and 9). 

Incubate the slides m a mixture of avidin—peroxidase conjugate diluted 1.100 and alkaline phosphatase-conjugated antidigoxigenin diluted 1 600 m TBT, in practice, 1 pL of antibody and 6 pL of avidin are added to 600 pL of TBT. 

Incubate the slides in alkaline phosphatase conjugated antidigoxigenin diluted 1 600 in TBT. 

At the time of writing, the antidigoxigenin antibody is available conjugated only to alkaline phosphatase. Peroxidase detection cannot therefore be used with this method. The amplified methods, however, allow greater flexibility. 

Fig. 4. Schematic diagram of the steps in the automated PCR/OLA procedure performed with a robotic workstation. The assay contains three steps (1) DNA target amplification (2) analysis of target nucleotide sequences with biotin (B)-labeled and digoxigenin (D)-labeled oligonucleotide probes and T4 DNA ligase (L) and (3) capture of the biotin-labeled probes on streptavidin (SA)-coated microtiter wells and analysis for covalently linked digoxigenin by using an ELISA procedure with alkaline phosphatase (AP)-conjugated antidigoxigenin (aD) antibodies and a substrate (S). Reprinted with the permission of Nickerson el al. (N2) and the Proc. Natl. Acad. Sci. (U.SA.).

Incubate the blots in approx 200 mL of antidigoxigenin solution for 30 min. At this point m the process the antibody/alkaline phosphatase conjugate binds to the digoxigenin-labeled probe that is bound to the membrane. 

Antidigoxigenin antibody/alkaline phosphatase conjugate (available from Boehringer) A1/5000 dilution in Buffer 1 should be made immediately before use (Section 3.2., step 4). 30 mL are required for one 10 x 18 cm filter, 70 mL for 10 filters. 

Enzyme-mediated reporter systems work by catalyzing the precipitation of a visible product at the site of probe hybridization. Antidigoxigenin antibodies are available conjugated to either alkaline phosphatase or horseradish peroxidase (HRP). In this chapter, the enzyme HRP is used to catalyze the oxidation of diarainobenzidine (DAB) to produce a brown precipitate at the site of in situ hybridization (Section 3.3.). 

This chapter describes the use of digoxigenin-labeled probes to detect mRNA transcripts in tissue sections. The sites of hybridization of the probe are visualized using an antidigoxigenin antibody alkaline phosphatase conjugate, and a colorimetric reaction. 

This chapter describes a nonradioactive method for the localization of mRNA in whole mouse embryos. It employs riboprobes labeled with digoxigenin, a steroid-like moiety not found in animal tissue. Digoxigenin-containing probe is visualized with a conjugate of antidigoxigenin Fab and alkaline phosphatase and colorimetric staining. The results are visualized in three dimensions, hence subtle patterns can be visualized without laborious sectioning. 

At this point begin preadsorbing the antidigoxigenin Fab/alkaline phosphatase conjugate see Section 2.). 

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