64 BIOTECHNOLOGY
Fig. 3. IR fluorescence signals are directly proportional to the amount of antigen, across a very wide linear range. The experiment from Fig. 1 was repeated using IR-labelled antibodies detected with an Odyssey infrared imaging system
saturated. When many silver grains are activated, each additional photon is statistically unlikely to strike an unactivated grain. Photons that do not activate a silver grain are not recorded as optical density, causing dramatic under- representation of strong signals. Tis is called ‘high intensity reciprocity failure’ and is demonstrated in the ‘shoulder’ region of the graph in Fig. 2. Artifacts from the film
development process can obscure image data. Bands can appear blurry if the blot or film shifts during handling. Strong signals can blow out and bleed into other bands. Mechanical damage from the developer and even static electricity can leave marks on film. Such artifacts can make a blot difficult to interpret, limiting its analytical usefulness. Office scanners are often used to digitise film for analysis. However, scanners cannot accurately reproduce original image data, and the dynamic range of scanned images is 2.6 times lower than CCD images. Among other problems, scanners truncate large signal peaks to limit output optical density to a factory preset range. Tis compromises data integrity, because not all peaks are recorded the same way.
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Unlike chemiluminescent methods, IR fluorophores provide stable and reproducible signal over time. Reproducibility is enhanced, because timing no longer affects results. Properly stored IR blots are stable for 18 months or more, and can be re- imaged later with the same results. Te linear, proportional
relationship between IR fluorescent signal and amount of antigen (Fig. 3) enables accurate comparison and protein quantification. Moreover, IR fluorescence technology provides over six logs of linear dynamic range (LDR). By contrast, manufacturers claim an LDR of about 100-fold for CCD imagers and closer to 15-fold has been observed. Film’s LDR varies from eight-fold to roughly 10-fold. With the wide LDR from IR imaging, one digital image contains the information provided by all possible film or CCD exposures, with much deeper analytical detail.
Fewer manual steps IR blots are imaged directly to a digital medium, which saves time and eliminates variability caused by substrate incubation, film exposure, and digital scanning of film. Tis streamlined workflow leads to greater data reproducibility.
Finally, IR technology
allows multiple targets to be detected on the same blot using spectrally-distinct fluorophores. Multiplex detection improves quantification accuracy by allowing one channel to be used for normalisation and eliminating the error-prone stripping and re-probing process. IR technology is an excellent
alternative to film exposure, providing reproducible results with stable, quantitative Western blot signals and a streamlined workflow.
For more information ✔ at
www.scientistlive.com/eurolab
Fig. 4. This graph encompasses data from all possible film exposures
REFERENCES: 1
Lundstrom K. (2009) An
overview on GPCRs and drug discovery: structure- based drug design and strutural biology on GPCRs. Methods Mol Biol. 552:51-66. 2
Stoddart et al. (2015)
Sam Egel is with LI-COR Biosciences.
www.licor.com
Application of BRET to monitor ligand binding to GPCRs. Nat. Methods 12(7): 661-663.
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