Multicolor Fluorescent Western Detection

Get more data from every experiment with multicolor fluorescent Western blotting

Advansta offers a complete line of products for fluorescent Western blotting. Our WesternBright MCF kit for multicolor fluorescent detection outperforms other fluorescent blotting systems, providing higher sensitivity and a shorter time to results since WesternBright MCF blots do not have to dry before imaging can be conducted. If you are new to fluorescent blotting, the WesternBright MCF protocol is easily adapted from the chemiluminescent protocol you are using now. Contact Advansta for more information.

Multicolor fluorescent detection of Western blots (Figure 1) allows you to detect more than one protein on a single blot without needing to strip and re-probe the blot. If you are interested in assaying more than one protein in your sample, fluorescent detection can save you substantial time and money. You can detect your protein of interest at the same time as a loading control for more quantitative data, or assay the phosphorylated and non-phosphorylated isoforms of a protein simultaneously (Figure 2). Additionally, fluorescent detection does not require film, saving the expense of developing X-ray film associated with film-detection of chemiluminescent blots.

Figure 1. The principle of two-color fluorescent Western detection. Using secondary antibodies labeled with different fluorophores, two proteins can be detected on a single blot by controlling excitation and detection channels. WesternBright conjugates incorporate the phycobiliproteins allophycocyanin (APC) and R-phycoerythrin (RPE), extremely bright fluorescent proteins from cyanobacteria and eukaryotic algae.

Figure 2. Simultaneous detection of EGFR and phospho-EGFR with WesternBright MCF. The increase in phosphorylation of EGFR in response to EGF was detected. Chemiluminescence detection of the same experiment would require stripping and reprobing the blot, which can introduce errors due to protein loss, or incomplete stripping of primary antibody. Lysates from A431 cells (lane 1) and from A431 cells treated with EGF (lane 2) were blotted and EGFR detected in the green channel (b) or phospho-EGFR detected in the red channel (c). The two channels are superimposed in (a). Lane 3: molecular weight markers.

Multicolor fluorescent detection

The WesternBright MCF kit contains all the necessary materials for carry out two-color fluorescent Westerns, including low-fluroescence PVDF transfer membranes, fluorescently-labeled secondary antibodies. The antibodies included in WesternBright MCF are labeled with phycobiliproteins, extremely bright fluroophores than make WesternBright MCF more sensitive than other fluorescent detection systems

Figure 3. WesternBright conjugates provide a brighter signal than the ECL Plex™ Western blotting system. Duplicate Western blots containing samples of AFP and CEA proteins were treated identically, and probed with the same mixture of mouse anti-AFP and rabbit anti-CEA primary antibodies. One blot was then stained with WesternBright conjugates and the other with an identical concentration of Cy3 anti-mouse and Cy5 anti-rabbit antibodies following the protocol recommended for ECL Plex. Under identical imaging conditions, WesternBright MCF provides a brighter signal, and 10x greater sensitivity.