ANTI-DIGOXIGENIN AP-CONJUGATE

General description

Digoxigenin is a hapten, useful in labeling and detection of nucleic acids. This product contains Fab fragments from polyclonal anti-digoxigenin antibodies, conjugated to alkaline phosphatase. Anti-Digoxigenin-AP, Fab fragments are useful for the detection of digoxigenin-labeled compounds.

Specificity

The polyclonal antibody from sheep is specific to digoxigenin and digoxin and shows no cross-reactivity with other steroids, such as human estrogens and androgens.
Heat inactivation: yes

Application

Use Anti-Digoxigenin-AP, Fab fragments for the detection of digoxigenin-labeled compounds using:

• cDNA array
• Colony/plaque hybridization
• Dot blot
• ELISA
• Gel shift assay
• Immunohistocytochemistry
• In situ hybridization
• Nonradioactive DNA sequencing blot
• Northern blot
• RNase protection assay
• Southern blot
• Western blot
• Fluorescent in situ hybridization
• Section in situ hybridization and whole mount in situ hybridization
• Electrophoretic mobility shift assay

Preparation Note

After immunization with digoxigenin, sheep IgG was purified by ion-exchange chromatography, and the specific IgG was isolated by immunosorption. The Fab fragments obtained by papain digestion were purified by gel filtration, conjugated to the specific label, and stabilized in buffer.
Working concentration: Working concentration of conjugate will depend on the application and substrate. The following concentrations should be taken as a guideline:

• Dot blot: 150 mU/ml
• ELISA: 150 to 300 mU/ml
• Immunohistocytochemistry: 250 to 500 mU/ml
• In situ hybridization: 1.5 to 7.5 U/ml
• Southern blot: 150 mU/ml
• Western blot: 250 to 500 mU/ml

Working solution: Northern blot, Southern blot
100 mM Maleic acid, 150 mM NaCl, pH 7.5.
Western blot
50 mM Tris-HCl, 150 mM NaCl, pH 7.5
other applications
100 mM Tris-HCl, 150 mM NaCl, pH 7.5

1% Blocking reagent (w/v), 1 to 5% heat inactivated fetal calf serum (v/v) or sheep normal serum can be used for reduction of unspecific binding.
Storage conditions (working solution): Diluted conjugate is stable at 2 to 8 °C for 12 hours.
Always prepare freshly!

Analysis Note

• Cross reactivity to digitoxin and digitoxigenin: <1 %
• No cross reactivity with other human estrogen or androgen steroids, e.g. estradiol or testosterone
• Cross reactivity with digoxin: not known
• Conjugate does not bind to itself at all
• Normally one molecule of the conjugate binds to one molecule digoxigenin, although ther are two possible binding sites for digoxigenin
• Nonspecific binding to RNA is not expected

Other Notes

For life science research only. Not for use in diagnostic procedures.