User Ratings

Easy to Follow Achieved Expected Results Overall Rating Add your comments

Chemiluminescent Detection of Nonisotopic Probes

Heather Perry‐O'Keefe¹, Carol M. Kissinger¹

¹Millipore 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

Are you the author of this protocol? Login or register and return to this page.

Abstract

With recent advances in techniques for detecting chemiluminescent substrates, hybridization with nonisotopic rather than radiolabeled probes is becoming more common. In the Basic Protocol, 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 Support Protocol 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 Alternate Protocol 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

Unit Introduction
Basic Protocol: Chemiluminescent Detection of Biotinylated Probes
Alternate Protocol: Chemiluminescent Detection of Digoxigenin-Labeled Probes
Support Protocol: Calibrating an Ultraviolet Light Source
Reagents and Solutions
Commentary
Bibliography
Figures
Tables

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Materials

Basic Protocol: Chemiluminescent Detection of Biotinylated Probes

Materials

Uncharged nylon membrane blotted via neutral transfer with DNA (UNIT 2.9A) or RNA (UNIT 4.9)
Biotinylated probes (UNIT 3.18)
Blocking solution
Wash buffers I and II
1 mg/ml streptavidin (see reagents and solutions)
0.38 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 (support protocol)
Heat-sealable hybridization bags

Additional reagents and equipment for Southern and northern hybridizations (UNITS 2.9A & 4.9)

Table 3.19.2 Chemiluminescent Substrates for Detection of Nonisotopic Probes^a

Dioxetane substrate	Buffer	Source^c

Lumigen-PPD^b (0.33 mM)	2-amino-2-methyl-1-propanol (pH 9.6)/0.88 mM MgCl₂/750 mM CTAB/1.13 mM fluorescein surfactant	BM, GB, LU, MI, NEB
Lumi-Phos 530^a (0.33 mM)	2-amino-2-methyl-1-propanol (pH 9.6)/0.88 mM MgCl₂/750 mM CTAB/1.13 mM fluorescein surfactant	BM, GB, LU, MI, NEB
AMPPD (0.25 mM)	1 mM DEA/1 mM MgCl₂, pH 10	TR
CSPD (0.25 mM)	1 mM DEA/1 mM MgCl₂, pH 10	TR

^a Abbreviations: AMPPD, disodium 3-(4-methoxyspiro1,2-dioxetane-3,2-tricyclo[3.3.1.1^3,7] 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.

^b Lumi-Phos 530 has a fluorescence enhancer; Lumigen-PPD does not.

^c Abbreviations: 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 4.

Support Protocol: Calibrating an Ultraviolet Light Source

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

Looking for materials? Suppliers Guide

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 3.19.1

Chemiluminescence reaction. The enzyme alkaline phosphatase cleaves a phosphate group off the chemiluminescent substrate. The intermediate is unstable and quickly decomposes, emitting light in the process. Reprinted with permission from Millipore.

View Image
Figure 3.19.2

Hybridization bag spout. After a buffer has been added to the bag, the spout is folded over and held closed with a bulldog clamp or several paper clips. It is important to leave sufficient room for the membrane to float free in solution and the bag should be free of trapped air to ensure maximum contact between the reagents and the membrane.

View Image

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.