While developing an antibody array for analysis of biomarkers in serum samples, we needed to confirm the performance of the antibodies that we used by an independent method. We evaluated the new instrument from Alpha Innotech designed for imaging and analysis of multi-color fluorescent Western blots in a quantitative manner. On the outside, the instrument looks similar to a standard gel documentation box. However, the instrument provides many more imaging choices. There is a dual wavelength UV transilluminator and a white light folding table for stained gels. The impressive features are the additional epi-illumination light sources. Our version included four additional light sources: epi UV, 475 nm blue, 534 nm green and 632 nm red lights. The light sources are configured to provide a very uniform narrow band illumination over the imaging area.
A technical representative installed the instrument and provided on-site training. The instrument really impressed us and performed extremely well from day one. We did not have previous experience staining Western blots with fluorescently labeled antibodies, so at this point, we expected a few “funny looking pictures” and not much else extraordinary from the instrument. However, FluorChem Q made the transition from other common methods to the fluorescent technique very easy and straightforward. In fact, after only a couple of experiments, we realized that fluorescent staining of Western blots is easier and quicker than a colorimetric or chemiluminescent technique and the instrument played a pivotal role in the transition. The instrument helped us find new ways of improving and/or troubleshooting Westerns. For example, if at the end of an experiment, the sensitivity does not appear sufficient, since there is no substrate involved, one could go back and repeat an incubation of the same blot with an antibody and add more, if necessary, to increase the signal intensity. Multi-color imaging provided by the instrument allows us to simultaneously and independently detect two or even three different proteins, which saves a lot of time and improves the results.
Since the instrument exceeded our expectations for Western blots, we decided to evaluate it for other commonly performed techniques. We used the instrument to image agarose gels after separation of DNA fragments and had another pleasant surprise. We found that LED excitation lights are more convenient for the imaging of agarose gels than a UV-transilluminator. Many common dyes (e.g. SybrGreen, SybrGold, SybrSafe) can be excited with the 475 nm blue light just as well as with a UV transilluminatior. The benefit is that the background is lower and the image quality is better compared to the UV excitation. For example, those annoying particles that are often present in agarose and fluoresce in the UV light are invisible when exciting with the blue light; and the bands do not fade during illumination since dyes do not degrade under the blue light. One could still use the available UV transilluminator when required, and it works great too.
Since the instrument is totally flexible, one could choose any combination of an excitation light source and an emission filter. The instrument has a 6-position filter wheel, which allows up to 5 emission filters. One position needs to remain with no filter to be able to image chemiluminescent blots, which also works extremely well. Overall, the instrument exceeded our expectations with just about any application we tried with it.
Principal Scientist
Advansta Inc.