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Western Blots and RNAi

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Figure 1. Validation of siRNA knockdown in A431 cells by two-color quantitative Western Each siRNA target is detected in green. In red, tubulin was detected as used as a loading control to normalize for sample variation. Graph indicates % of target expression in siRNA-treated cells, relative to controls. In each blot, the marker is at the left, the negative control is in the center, and the siRNA is at the right.

Figure 2. Clear, detailed blot images of siRNA knockdown of ERK2 was performed in A431 cells. ERK2 detection is shown in green (primary antibody binds both ERK isoforms); tubulin loading control is shown in red. In this Western blot, the spatial resolution of ERK isoforms demonstrates that when ERK2 expression is lowered with siRNA treatment, ERK1 expression increases.

Quantitative Western blotting is an essential tool for every step in your RNAi studies:

  • Choosing an RNAi vehicle (siRNA or shRNA or other RNA-producing vector construct)
  • Confirming level of knockdown of the target gene
  • Determining the impact of target gene knockdown on a phenotype such as cell proliferation, cell migration, cell cycle, or cell signaling pathways

The Odyssey® CLx Infrared Imaging System provides accurate digital protein data for your RNAi studies.

  • Raw data can be normalized for increased accuracy using one of the two independent channels (colors) to monitor a control protein.
  • Stable fluorescent signal is directly proportional to amount of target for consistent data.
  • Wide linear range allows knockdown levels involving both weak and strong signals to be measured accurately in a single image without the bleed over of signal from adjacent "blow out" bands.
  • In-Cell Western™ assays also offer additional throughput for more complex studies, as well as exceptionally consistent data (Z'-factor).1 The ICW has recently been demonstrated as a powerful cellular assay for genome-wide RNAi screens. 2


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