Chemiluminescent Detection of Nonisotopic Probes

Heather Perry‐O'Keefe1, Carol M. Kissinger1

1 Millipore Corporation, Burlington, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.19
DOI:  10.1002/0471142727.mb0319s20
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

With recent advances in techniques for detecting chemiluminescent substrates, hybridization with nonisotopic rather than radiolabeled probes is becoming more common. In the , nylon membranes carrying transferred nucleic acids are prepared for hybridization with biotinylated probes by UV cross‐linking. This is a critical step in the procedure and the provides a detailed description of light‐source calibration. After hybridization, the target nucleic acid is detected through a series of steps that lead to an enzyme‐catalyzed light reaction. The describes chemiluminescent detection based upon antibody recognition of digoxigenin‐labeled probes. For both biotinylated and digoxigenin‐labeled probes, chemiluminescent detection is more sensitive than colorimetric detection and has the added advantage that the membrane can be used for multiple film exposures, then stripped and redetected with different probes.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Chemiluminescent Detection of Biotinylated Probes
  • Alternate Protocol 1: Chemiluminescent Detection of Digoxigenin‐Labeled Probes
  • Support Protocol 1: Calibrating an Ultraviolet Light Source
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Chemiluminescent Detection of Biotinylated Probes

  Materials
  • Uncharged nylon membrane blotted via neutral transfer with DNA (unit 2.9) or RNA (unit 4.9)
  • Biotinylated probes (unit 3.18)
  • recipeBlocking solution
  • Wash buffers recipeI and recipeII
  • recipe1 mg/ml streptavidin (see reagents and solutions)
  • recipe0.38 mg/ml biotinylated alkaline phosphatase
  • Chemiluminescent dioxetane substrate (Table 3.19.2)
  • Substrate buffer, pH 9.6
  • Blotting paper (Whatman 3MM or equivalent)
  • Calibrated UV source ( protocol 3support protocol)
  • Heat‐sealable hybridization bags
  • Additional reagents and equipment for Southern and northern hybridizations (units 2.9 & 4.9)
    Table 3.9.2   Materials   Chemiluminescent Substrates for Detection of Nonisotopic Probes b   Chemiluminescent Substrates for Detection of Nonisotopic Probes

    Dioxetane substrate Buffer Source d
    Lumigen‐PPD c (0.33 mM) 2‐amino‐2‐methyl‐1‐propanol (pH 9.6)/0.88 mM MgCl 2/750 mM CTAB/1.13 mM fluorescein surfactant BM, GB, LU, MI, NEB
    Lumi‐Phos 530 b (0.33 mM) 2‐amino‐2‐methyl‐1‐propanol (pH 9.6)/0.88 mM MgCl 2/750 mM CTAB/1.13 mM fluorescein surfactant BM, GB, LU, MI, NEB
    AMPPD (0.25 mM) 1 mM DEA/1 mM MgCl 2, pH 10 TR
    CSPD (0.25 mM) 1 mM DEA/1 mM MgCl 2, pH 10 TR

     bAbbreviations: AMPPD, disodium 3‐(4‐methoxyspiro{1,2‐dioxetane‐3,2‐tricyclo[3.3.1.13,7] decan}‐4‐yl)phenyl phosphate; CSPD, AMPPD with substituted chlorine group on adamantine chain; CTAB, cetyltrimethylammonium bromide; DEA, diethanolamine; Lumigen‐PPD and Lumi‐Phos 530: 4‐methoxy‐4‐(3‐phosphate phenyl)‐spiro‐(1,2,‐dioxetane‐3,2‐adamantine), disodium salt.
     cLumi‐Phos 530 has a fluorescence enhancer; Lumigen‐PPD does not.
     dAbbreviations: BM, Boehringer Mannheim; GB, GIBCO/BRL; LU, Lumigen; MI, Millipore; NEB, New England Biolabs; TR, Tropix. Addresses and phone numbers of suppliers are provided in appendix 44.

Alternate Protocol 1: Chemiluminescent Detection of Digoxigenin‐Labeled Probes

  Additional Materials
  • UV source: transilluminator, hand‐held UV lamp, homemade box with germicidal bulbs, or cross‐linker (e.g., Stratalinker, Stratagene #400071)
  • Radiometer and radiometer sensor
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Beck, S. and Koster, H. 1990. Applications of dioxetane chemiluminescent probes to molecular biology. Anal. Chem. 62:2258‐2270.
   Creasey, A., D'Angio, L., Dunne, T.S., Kissinger, C., O'Keefe, T., Perry‐O'Keefe, H., Moran, L.S., Roskey, M., Schildkraut, I., Sears, L.E., and Slatko, B. 1991. Application of a novel chemiluminescence‐based DNA detection method to single vector and multiplex DNA sequencing. BioTechniques 11:102‐109.
   Schaap, A.P., Handley, R.S., and Giri, B.P. 1987. Chemical and enzymatic triggering of 1,2‐dioxetanes. 1:Aryl esterase‐catalyzed chemiluminescence from a naphthyl‐acetate substituted dioxetane. Tetrahedron Lett. 28:935‐938.
Key Reference
   Beck, S. and Koster, H. 1990. See above.
  Reviews the chemistry behind the techniques described and discusses the applications for this methodology.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library