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Fluorophore probe coupled to an antibody
The choice of fluorophore probes depends on the following important criteria:
A. Instrument. For example, light source, filter, detection system.
B. The requirement for the degree of color discrimination of the probe in multiple markers . For example, the difference between rhodamine-X (RRX) and Texas Red (TR) fluorescein is significantly different from that of tetramethylrhodamine (TRITC) or Cy3.
C. Required sensitivity. For example, Cy3 and Cy5 are brighter than other fluorophore probes.
Aminomethylcoumarin Acetate (AMCA)
The coupled AMCA absorbs light at a wavelength of up to 350 nm and fluoresces at 450 nm. For fluorescence microscopy, AMCA can be excited with a mercury lamp and viewed with a UV filter. Since AMCA's signal is relatively weak, AMCA is not recommended for single-label experiments. The fluorescence wavelengths of AMCA and fluorescein have only a small overlap range, and there is little or no overlap with the fluorophore that emits long-wavelength fluorescence, so it is most commonly used in multi-label experiments, such as immunofluorescence microscopy and flow cytometry. instrument. Since the human eye does not detect blue fluorescence well, in multi-labeled experiments, AMCA-coupled secondary antibodies should be used to detect large amounts of antigen. AMCA is quenched as fast as fluorescein and can be alleviated with anti-quenching agents. If used in flow cytometry, AMCA can be excited with mercury lamps or water-cooled argon lamps because they emit light in the ultraviolet region of the spectrum.
Fluorescein Isothiocyanate (FITC)
The coupled fluorescein group absorbs a maximum wavelength of 492 nm and emits a maximum wavelength of 520 nm. Since FITC has been used for a long time and the yield is large, FITC is widely used. The biggest disadvantage of fluorescein is that it is quenched quickly and therefore used with anti-quenching agents. DTAF is a derivative of fluorescein with the same excitation and emission wavelengths as FITC. When coupled with streptavidin, DMAAF is preferred because FITC is not used because of the significant difference in fluorescence intensity.
Cyanine dyes ( Cy2, Cy3, Cy5)
The Cy2 coupling group has an excitation wavelength of 492 nm and emits green visible light having a wavelength of 510 nm. Cy2 and FITC use the same filter. Since Cy2 is more stable than FITC under light. Avoid using a sealer containing phosphorylated phenylenediamine because this anti-quenching agent reacts with Cy2, resulting in weak fluorescence and diffusion after storage of the stained sheet.
Cy3 and Cy5 are brighter, more stable, and have a weaker background than other fluorophore probes. The maximum wavelength of the excitation light of the Cy3 coupling group is 550 nm, and the strongest emission light is 570 nm. Since the excitation and emission wavelengths are very close to TRITC, in fluorescence microscopy, the same filter as TRITC can be used.
Cy3 can be excited to 50% light intensity in an argon lamp ( 514 nm or 528 nm), and about 75% in a xenon lamp (543 nm) or a mercury lamp (546 nm). Cy3 can be double labeled with fluorescein. Cy3 can also be multi-labeled with Cy5 in confocal microscopy experiments.
The excitation wavelength of the Cy5 coupling group is 650 nm at the maximum and the emission wavelength is 670 nm at the maximum. They were excited to 98% of fluorescence under a helium argon lamp (647 nm) and 63% under a xenon lamp (633 nm). Cy5 can be used in multi-label experiments with many other fluorophores. Since its maximum emission wavelength is 670 nm, Cy5 is difficult to observe with the naked eye, and it cannot be excited by a mercury lamp. Confocal microscopy with suitable excitation and far-infrared detectors is typically used when viewing Cy5. An anti-quenching agent should be added to the aqueous phase sealer. Cy5 is not recommended when using a conventional surface fluorescence microscope.
Tetramethyl Rhodamin Isothiocyanate ( TRITC)
Rhodamine Red-X (RRX), Texas Red (TR)
These rhodamine derivative coupling groups have different absorption wavelengths (550, 570, 596 nm) and maximum emission wavelengths (570, 590, 620 nm). Although TRITC is often used in conjunction with FITC in double labeling experiments, the use of RRX and TR results in better color discrimination. RRX is especially useful when using a laser confocal scanning microscope equipped with a helium argon lamp for triple labeling. It can be used with Cy2 (or FITC) and Cy5 because the emission wavelength of RRX is between Cy2 and Cy5, and this Both cover very little. The argon lamp excitation light is 488 nm, 598 nm and 647 nm, which are the ideal excitation wavelengths of Cy2 (FITC), RRX and Cy5, respectively. Because FITC and PE can be excited by the 488 nm wavelength of the argon lamp, FITC is used as a double label in the flow cytometer, and the other coupling element with phycoerythrin (PE) is better than rhodamine.
Affinity- chromatized antibodies against HRP and its couplings can be used to detect HRP or amplify the effect of containing HRP reagents. In immunostaining of mammalian cells, since the anti-HRP antibody does not recognize an endogenous peroxidase-like enzyme, the advantage of this antibody is that it can reduce the background.