Molecular Techniques and Methods

Chemiluminescent Detection of DIG-labled Probe

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

Genius Buffer 1 (1 L)
100 mM Tris-HCl(pH7.5) ----------------------- 50 ml of 2 M Tris-HCl
150 mM NaCl ---------------------------------- 30 ml of 5 M NaCl
0.3% Tween-20 -------------------------------- 3 ml of Tween-20
DW ------------------------------------------------ 917 ml
  • Filter through a 0.45 um membrane.


    Genius Buffer 2 (10 ml)
    2% Blocking reagent -------------------------- 10% Blocking reagent - 2 ml
    Genius buffer 1 ------------------------------ 8 ml
  • Stable for 2 weeks at 4oC.


    Genius Buffer 3 (500 ml)
    100 mM Tris-HCl (pH9.5) ---------------------- 2 M Tris-HCl - 25 ml
    100 mM NaCl ---------------------------------- 5 M NaCl - 10 ml
    50 mM MgCl2 ---------------------------------- 1 M MgCl2 - 25 ml
    DW ------------------------------------------- 440 ml
  • Filter through a 0.45 um membrane.


    Anti-DIG-alkaline phosphatase
    Anti-digoxigenin conjugated to alkaline phosphatase


    Lumi-Phos 530
    0.33 mM Lumigen PPD [4-methoxy-4-(3-phosphatephenyl)-spiro(1,2-dioxetane-3,2'-ademantane) disodium salt]
    750 mM 2-amino-a-methyl-1-propanol buffer (pH9.6)
    0.88 mM MgCl2
    1.13 mM cetyltrimethyl-ammonium bromide
    0.035 mM fluorescein surfactant




    PROCEDURES

    Perform all incubations at room temperature.

    1. After hybridization and post-hybridization wash, equilibrate the membrane in Genius Buffer 1 for 1 min.

    2. Block the membrane in Genius Buffer 2 in the bag for 30-60 minutes by gently agitating.

    3. Prepare the antibody solution as follow.
    Dilute the anti-DIG-alkaline phosphatase 1:5000-1:10,000 in Genius Buffer 2 (150 mU/ml).
    Ex) 2 ul anti-DIG-alkaline phosphatase in 10 ml Genius Buffer 2
  • This antibody solution is stable for 12 hrs at 4oC.

    4. Incubate the membrane for 30 min, room temperature in the antibody solution in the bag.

    5. Transfer the membrane to a dish.

    6. Wash the membrane in 100 ml Genius Buffer 1 for 15 min. 2x.

    7. Equilibrate the membrane in 20 ml Genius Buffer 3 for 2 min.

    8. Place the membrane between two sheets of acetate (plastic page protectors).
    Apply 0.5 ml Lumi-Phos 530/ 100 cm2 membrane.
    Rock the membrane gently to distribute the reagent over the surface.

    9. Expose membrane to X-ray film for 1 hr to over night at room temperature.




  • NOTES




    KIT INFORMATION



    REFERENCES


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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