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Tip: Make sure you detect your sample and your loading controls in the linear range.
Tip: Determine protein concentration with an assay.
Tip: Monitor migration with a pre-stained molecular weight marker.
Tip: Optimize blocking conditions by comparing a minimum of three different blocking buffers to find the best blocker to fit your experimental needs.
Tip: Validate that your antibodies recognize the correct protein target and don’t cause non-specific binding before you begin.
Tip: Clean incubation boxes with methanol before you use them to prevent contamination.
Tip: Eliminate cross-reactivity on multiplex Westerns with highly cross-adsorbed secondary antibodies, like the IRDye® Secondary Antibodies.
Tip: Use freshly-made buffers to prevent contamination.
Tip: Use a detection system with a wide linear dynamic range, like the Odyssey® Imaging Systems.
Tip: Let Empiria Studio Software handle the background subtraction method.