To properly quantitate proteins in Western blotting, it is necessary to control for variations in total protein from lane to lane as well as for variations in the volumes of samples added to the wells.
This is usually accomplished by quantitating levels of an unregulated, or “housekeeping”protein, in addition to the target protein itself, by stripping the membrane of the antibody used for detection of the target protein and reprobing with a different antibody directed against the housekeeping protein. The stripping procedure, however, carries a risk of uneven loss of protein from the membrane surface. With fluorescence-based Amersham ECL Plex, stripping is no longer required, due to the possibility of multiplexed detection.
Multiplexed Detection in Western Blotting
Here, the activation of the intracellular signaling molecule, ERK 1/2 , was studied in lysates of wildtype and knockout fibroblasts treated with fibroblast growth factor-2 (FGF-2). The lysates were separated by PAGE, and after blotting, the membrane was simultaneously probed with mouse anti-ERK 1/2 and rabbit anti-GAPDH, followed by Amersham ECL Plex anti-mouse Cy5 (red light emission) and Amersham ECL Plex anti-rabbit Cy3 (green light emission).
By detecting two proteins on the same blot, protein expression can be quantitated relative to a housekeeping protein such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data courtesy of Dr. Jin-Ping Li and Dr. Juan Jia, Department of Medical Biochemistry and Microbiology, Uppsala, Sweden.
Although protein quantitation analysis indicated that a similar amount of total protein was loaded in each lane, the intensity of the signals emitted due to detection of the housekeeping protein, GAPDH, clearly showed that this was not the case. Without relating to the GAPDH levels, no significant pattern of ERK1/2 activation related to wildtype and knockout cells was seen. However, when normalized against GAPDH signals, ERK 1/2 levels were shown to increase in knockout cells exposed to FGF-2.
Sensitive Detection of Low Abundance Proteins
The need to detect low abundance proteins, for example in the study of signaling cascades, has increased the requirements for sensitivity and linear dynamic range when using techniques such as gel- and/or blot-based quantitative analysis. A wide linear dynamic range is particularly important for applications where weak and strong signals are compared in the same experimental series. A two-fold dilution series of transferrin, for example, was detected using Amersham ECL Prime, and analyzed using CCD camera-based ImageQuant LAS 4000 mini. The analysis was both highly sensitive and quantitative across a linear dynamic range covering several orders of magnitude.
Sensitive detection over a wide dynamic range using Amersham ECL Prime to detect transferrin on a Western blot.