D

Figure 11 (A) Structure of Eu3+ cryptate. In Eu3+ cryptate (EuK) the Eu3+ is tightly bound within the trisbipyridine cryptate cage that protects Eu3+ from quenching, enhances Eu3+ fluorescence, and provides convenient linkers to attach biomolecules. (B) Structure of Eu3+ chelate. (C) and (D) Structure of Tb3+ chelates.

maximally at 613 nm to longer wavelength acceptor molecules such as allophyco-cyanins XL665 or Cy5 and from Tb chelates emiting at 492 and 545 nm to a variety of acceptors such as tetramethylrhodamine, xanthine dyes, or fluorescein [25,26,28]. Some of the generic reagents available include Eu3+ or Tb3+ labeled antihuman, rabbit or mouse IgG, anti-GST or anti-PY antibody, protein G and streptavidin, and APC labeled streptavidin (Advant).

1. Instrumentation

The dual-wavelength time-resolved fluorometer that can measure time-resolved fluorescence and the ratio of donor fluorescence and acceptor fluorescence is the Discovery HTRF microplate analyzer (Packard) [28]. The schematic for the Discovery is given in Ref. 28. The Discovery simultaneously measures the emission of (Eu)K at 620 nm and the XL665 at 665 nm and the ratio is calculated. The other fluorometers that can measure TRF at two wavelengths one after the other are the 1420 Victor plate reader (EG&G Wallac, Turku, Finland), the Analyst plate reader (LJL Biosystems), and the PolarStar (BMG Technologies). The Victor 1420 schematic diagram of optics is given in Figure 12.

Aperture slide Emission filter

Aperture slide Emission filter

Mirror Diode Flash Lena bloc* block excitation Mock filter slide

Figure 12 Optical pathway of Victor 1420. Excitation light from UV Xenon flash tube (1500 V) is focused through a filter, conventional lenses, and a dichroic mirror into the sample, and the emitted light passes through a narrow band filter into a R1527 PMT for measurement. The dual window TRF option allows us to record two separate windows in one flash cycle. (Courtesy of Wallac.)

Mirror Diode Flash Lena bloc* block excitation Mock filter slide

Figure 12 Optical pathway of Victor 1420. Excitation light from UV Xenon flash tube (1500 V) is focused through a filter, conventional lenses, and a dichroic mirror into the sample, and the emitted light passes through a narrow band filter into a R1527 PMT for measurement. The dual window TRF option allows us to record two separate windows in one flash cycle. (Courtesy of Wallac.)

Was this article helpful?

0 0

Post a comment