Fluorescent Western Calibration Blot
A convenient way to demonstrate the performance of your imager
This pre-processed Western blot was developed as a tool to verify the performance of fluorescence imaging systems, including laser scanners and CCD-based instruments. Antibodies used on the blot fluoresce in the red, green, and blue fluorescent channels.
When the blot image is acquired with an instrument equipped with LEDs or lasers to excite Cy2, Cy3, and Cy5 (or equivalent dyes), the membrane produces a composite image similar to Figure 1. When placed on the surface with the long side vertical and the cut notch in the upper right corner, the working surface containing fluorescent samples is facing up towards the user.
Figure 1. Simultaneous detection of three fluorescently labeled antibodies on one blot. The standardization blot is ready to use, bound with antibodies that can be detected in three fluorescent channels. The standardization blot contains HeLa cell lysate (1, 2, 3 and 5 µg) spiked with 200 ng of human transferrin per lane. Transferrin is detected in the red channel (b), tubulin is detected in the green channel (c) and GAPDH is detected in the blue channel (d). The three channels are superimposed in (a). The first lane contains molecular weight markers that may be detected in the red channel.
A pre-processed fluorescent Western blot
Proteins were separated by polyacrylamide gel electrophoresis followed by transfer to a low-fluorescent PVDF membrane. Two proteins, tubulin and transferrin were detected by primary antibodies made in rat and rabbit hosts, followed by secondary antibodies labeled with different fluorescent dyes. Next, GAPDH was detected by a goat primary antibody directly labeled with SpectraDye Antibody Labeling Kit-Dye 490. The included background quenching sheet with low auto-fluorescence should be used for blot imaging to greatly reduce general background, especially in the blue channel.
Proteins were loaded onto the gel as indicated in the table.
|HeLa Lysate (ug)||1||2||3||5|