Appendix A: Working Solutions for Immunodetection of Southern Blot
| Working Solution | Composition / Preparation | Storage / stability | Use |
|---|---|---|---|
| Maleic acid buffer | 0.1 M Maleic acid, 0.15 M NaCl; adjust with NaOH (solid) to pH 7.5. (20°C) | 15–25°C, stable | Dilution of Blocking solution |
| Washing buffer | 0.1 M Maleic acid, 0.15 M NaCI pH 7.5 (20°C), 0.3% (v/v) Tween 20 | 15–25°C, stable | Removal of unbound antibody |
| Blocking solution | Prepare a 1 x working solution by diluting 10x Blocking reagent 1:10 with Maleic acid buffer. | prepare fresh | Blocking of unspecific binding sites |
| Antibody solution | Centrifuge Anti-Digoxigenin-AP for 5 min at 10,000 rpm in the original vial before each use, and pipet the necessary amount carefully from the surface. Dilute anti-digoxigenin-AP 1:5000 (150 mU/ml) in the Blocking solution. (1 µl to 5 ml) | 2 hours at 4°C | Binding to the DIG-labeled probe |
| Color substrate solution | Add 40 µl of NBT/BCIP stock solution (vial 5) to 2 ml of Detection buffer. Note: Store protected from light! | Prepare fresh | Visualization of antibody-binding |
| Detection buffer | 0.1 M Tris-HCI, 0.1 M NaCl, pH 9.5 (20°C) | 15–25°C | Adjustment to pH 9.5 |
| TE buffer | 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 | 15–25°C | Rinse membrane |
*DIG application manual for filter hybridization [Internet]. Darmstadt (Germany): Sigma-Aldrich; [accessed 2023 Feb 11]. Available from: https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/marketing/global/documents/199/985/dig-application-manual-for-filter-hybridisation-iris.pdf.
