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High quality fluorescent antibodies

FAQ - Fluorescent detection in immunological applications

Fluorescence has brought increased sensitivity, larger detection ranges and multiplex capacity unknown until now. Thus, from immuno-fluorescence to western blot, researchers are indeed now offered far more powerful techniques, but also confronted with totally new constraints: selection of non-crossreacting antibodies and intensity, photostability or narrowness of fluorochromes emission.

The following questions & answers give a brief insight into this subject.

Question: What can be recommended for flow cytometry?

Answer: FCM is the most robust immunological application. The extremely fast laser exposure allows any fluorochrome to be detected with no risk of bleaching, if it fits with laser and filters wavelengths. FITC, PE, APC and their respective tandems are the most commonly selected labels. In this technique where target multiplex detection is the rule, researchers are advised to select fluorescence conjugated primary antibodies, for example our selection of FCM validated primaries labelled with FITC, PE, PeRCP, PE-Cy7 or PerCP-CY5.5 or even Alexa…

For example: Anti beta2-microglobulin AF488

Find them: 
At www.tebu-bio.com/to/antibody: search your target name, then refine on application FCM and/or label of your choice.

Question: What about immuno-fluorescence and derivatives, such as confocal microscopy?

Answer: Due to the much longer exposure time, stable fluorochromes are needed to resist to photobleaching. Multiplex target detection is very common, but in this case targeted antigens can change for each assay, making selection of antibodies more critical. The best solution is to select highly cross-adsorbed secondary antibodies which will guarantee optimal primary discrimination, and a short selection of fluorescent labels. For example, DyLight 405, 488, 549, 649, 680 and 800 are compatible with most fluorescence microscopes and can be combined to constitute a reference panel. You will find these conjugated to the high titer cross-adsorbed Rockland secondaries.

Typical selection:

Find them:
At www.tebu-bio.com/to/secondaryantibody refine on host, target and cross-adsorption species, and label of your choice.

Question: Fluorescence microscopy technologies recently evolved to get better image resolution with devices such as Leica STED microscope. How do the fluorescence conjugated antibodies adapt to that evolution?

Answer: These new devices are much more demanding in terms of photo-bleaching resistance, stability of the fluorochromes... According to Leica, the best fluorochromes for STED are ATTO dyes due to their optical properties, compatible with sub-diffraction imaging.

Find them:
At www.tebu-bio.com/to/atto refine on host, target and cross-adsorption species, and choose between ATTO dyes (425, 488, 532, 550, 594, 647N and 655).

Question: I often encounter high back-ground issues with live-cell or animal imaging, what can I do to reduce this?

Answer: Many endogenous proteins such as haemoglobin show a high autofluorescence. You will avoid this interference by using near-infrared fluorochromes which can be easily discriminated from the back-ground. DyLight 680 & 800 conjugated antibodies will help.

e.g.

Find them:
At www.tebu-bio.com/to/secondaryantibody refine on labels in the near infrared range.

Question : How can fluorescence improve Western blot?

Answer: Fluorescence imaging systems allow detection of your proteins in western blot, providing a longer dynamic range read-out compared to chemiluminescence. This allows to detect both low and high concentrated proteins in same assay. Moreover the multi-color capacity of the readers allows a precise comparison of 2 proteins at a time. A perfect combination is offered by the use of DyLight 680 and 800 on the LI-COR Odyssey near infrared device and any other conventional instrumentation. A combination of products much selected by our customers:

DyLight fluorescent dye spectra

Find them:
At www.tebu-bio.com refine on labels "DyLight 680" or "DyLight 800".

Question : Is it possible to combine fluorescent detection with the TrueBlot® technology to generate publication-quality IP / Western blot data?

Answer: Fluorescent TrueBlot® enables such a combination. It brings the power and specificity of the original Trueblot technology with the versatility of fluorescent and near infra-red dyes for a variety of immunoassays including multiplexing detections.

Find them:
At www.tebu-bio.com refine on labels "Trueblot".