AMPPD

Recently, two major enzyme-catalyzed chemiluminescent reactions have become popular. These use either luminol as a substrate of peroxidase or 3-(2 -spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD) as a substrate of alkaline phosphatase (ALP). 

AMPPD is the best chemiluminescent substrate for detecting an ALP-labeled probe [2, 3], The enhanced sensitivity of the chemiluminescence based on the reaction of AMPPD with ALP depends on the enzymatic reaction time (Fig. 2), because the slow kinetics of the signal decay result in the accumulation... 

FIGURE 6. (a) The pH dependence of the Michaehs constant (ATm) for the alkahne-phosphatase-catalyzed decomposition of AMPPD in 0.05 M carbonate buffer at 37 °C (b) the same pH dependence for the mmover number (fccat)- Reprinted with permission from Reference 50. Copyright (1996) American Chemical Society... 

From this detailed kinetic analysis of the complex pH dependence for the chemiluminescence intensity we conclude that only the enzymatic dephosphorylation of AMPPD... 

Substrates for AP are, e.g.,AMPPD (disodium 3-(4-methoxyspi-ro 1 >2-dioxethane-3,2 -ticyclo[3.3.1.1 ] decan -4-yl)phenyl phosphate) or CSPD (disodium 3-(4-methoxyspiro l,2-dioxethane-3,2 -(5"-chloro)tricyclo [3.3.1.1] decan -4-yl)-phenyl phosphate). 

B 0.1 M Tris, pH 9.5,0.1 M NaCl, 5 mM MgCh (TNM buffer) C AMPPD stock solution (from the supplier)... 

Substitute Soln. B by the AMPPD dilution (0.2 ml/cm ) and incubate for 5 min. Drain the blot and wrap it with a solvent-tight foil. Incubate the wrapped membrane at 37 °C for 5 min, and then expose the blot face towards to the film in an X-ray film cassette for 15 min to 24 h or photograph it with a digital camera. 

Nitro-Block enhances light output from the dioxetane substrate in reactions using AMPPD, CSPD, or Lumigen-PPD concentrate. It is required for nitrocellulose and recommended for PVDF membranes. It is not needed for Lumi-Phos 530, AP reactions on nylon membranes, or HRP-based reactions on any type of membrane. Lumi-Phos 530 is not recommended for nitrocellulose membranes. 

Traditionally, the AMPPD substrate buffer has been a solution containing I mM MgCl2 and 50 mM sodium carbonate/bicarbonate, pH 9.6 (Gillespie and Hudspeth, 1991). The use of diethanolamine results in better light output (Western Light instructions Applied Biosystems). 

Dioxetane phosphate visualization solution Prepare 0.1 mg/ml AMPPD or CSPD (Applied Biosystems) or 0.1 mg/ml Lumigen-PPD (Lumigen) substrate in dioxetane phosphate substrate buffer (see recipe). Prepare just before use. Lumi-Phos 530 (Boehringer Mannheim or Lumigen) is a ready-to-use solution and can be applied directly to the membrane. 

This concentration (240 pM) of AMPPD substrate is the minimum recommended by Applied Biosystems. Ten-fold lower concentrations can be used but require longer exposures. 

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