Fluorescent Western standardization blot

Pre-processed three-color fluorescent blot

This pre-processed blot was developed to demonstrate the performance of fluorescence imaging systems, including laser scanners and CCD-based instruments. Three different proteins are detected with antibodies that fluoresce in the red, green, and blue fluorescent channels.

This blot contains three proteins (AFP, CEA and transferrin) at different ratios in each lane. The proteins were separated on a 12% polyacrylamide gel followed by transfer to a low-fluorescence PVDF membrane. The blot was then probed with a mixture of primary antibodies raised in mouse and rabbit, and stained with a mixture of secondary antibodies labeled with different fluorescent dyes. AFP is detected in the green channel, typically excited with a green laser or LED. CEA is detected in the red channel, typically excited with a red laser or LED. Transferrin is detected by a goat primary antibody directly labeled with a dye that can be detected in the blue channel, typically excited with a blue (argon) laser or blue LED. Transferrin is present at a constant amount in all lanes, in order to demonstrate the utility of using directly-labeled primary antibodies as loading controls