New Light On Fluorescent Antibodies

Wed, 01/06/2010 - 10:00

Extra-bright antibodies increase the specificity and sensitivity of any fluorescence-based procedure. They are stable over a wide pH range and are also highly ‘photo stable.’

Novus Biologicals now offer more than 1,150 antibodies conjugated to DyLight® fluorescent dyes.

As fluorescent imaging technology advances, there is a growing drive to achieve increasingly quantitative measurement of fluorescent signals.

Ultimately, this would permit determination of the absolute quantity of a target molecule within a cell or compartment using a fluorescent antibody technique.

A major obstacle to such a technological breakthrough is the fact that the fluorescent intensity of some dyes can be significantly affected by various cellular environmental factors. Cellular organelles show a wide variation in pH, ranging from as high as 8.0 in mitochondria, to as low as 4.5 in lysosomes.

Chen et al (Anal. Chem. 2008, 80(19); pp. 7437-7444) investigated how fluorescent intensity may be affected by intracellular factors. Of the eight dyes studied, Dylight 649, Cy5, Cy7 and Texas Red showed the greatest resistance to pH. Their fluorescent intensity remained stable over a pH range of 3 to 10. Dylight 649, Alexa647 and Alexa750 were the only compounds that did not show changes in intensity when injected into the cytoplasm of living cells. Fluorescein was the poorest performer in both cases.

Thus Dylight 649 was clearly superior, being the only compound in the top-performing group in both cases. This seems to be a very impressive indication of the exciting possibilities offered by Dylight-conjugated antibodies in the antibody catalog.

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