Fluorescent Western calibration blot

Ready-to-image three-color blot



Ready-to-image three-color blot

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

Additional information


1 each


  • The easiest way to demonstrate the performance of your imaging system
  • Quality-controlled, with lot-specific statistics provided for every blot
  • Both fluorescently labeled primary and secondary antibodies are used, to demonstrate different experimental designs, and the utility of loading controls

Fluorescent Western standardization blot
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 (3, 5, 10, and 15 µg) spiked with 100 ng of human transferrin per lane. Transferrin is detected in the red channel (b) and GAPDH is detected in both the green channel (c) and 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.

When acquired with an imager 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 right upper corner, the working surface containing fluorescent samples is facing up towards the user.

The kit contains a sample of a multi-color fluorescent Western blot for demonstrating the capabilities and comparison of fluorescent imagers. The membrane contains proteins immuno-stained with three antibodies labeled with different fluorescent dyes. Proteins were separated by polyacrylamide gel electrophoresis followed by transfer to a low-fluorescent PVDF membrane. Two proteins, GAPDH and Transferrin were detected by primary antibodies made in mouse 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.

Lane 1 2 3 4 5
Transferrin (ng) 100 100 100 100
HeLa Lysate (ug) 3 5 10 15
M.W. Marker +


Standardization Blot Flyer

StandardizationBlot MSDS

Advansta Catalog

Order Information

Reference Description Quantity
K-08001-007 Fluorescent Western Standardization Blot 1 each

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