Basic Workflow for Quantitative Western Blots

Protocol for Quantitative Western Blots

Choose a Normalization Strategy

Tip: Make sure you detect your sample and your loading controls in the linear range.

Western Blot Normalization Handbook

Prepare Samples

Tip: Determine protein concentration with an assay.

Perform Electrophoresis

Tip: Monitor migration with a pre-stained molecular weight marker.

Transfer to Membrane

Tip: Use the right membrane to minimize background, and validate whether your protein binds best to nitrocellulose or PVDF.

Protein Electrotransfer Methods Technical Note


Tip: Optimize blocking conditions by comparing a minimum of three different blocking buffers to find the best blocker to fit your experimental needs.

Odyssey Western Blot Blocker Optimization Technical Note

Incubate with Primary Antibodies

Tip: Validate that your antibodies recognize the correct protein target and don’t cause non-specific binding before you begin.

Get personalized publications report specific to your target


Tip: Clean incubation boxes with methanol before you use them to prevent contamination.

Incubate with Near-Infrared Fluorescent Secondary Antibodies

Tip: Eliminate cross-reactivity on multiplex Westerns with highly cross-adsorbed secondary antibodies, like the IRDye® Secondary Antibodies.

Western Blot and In-Cell Western Assay Detection using IRDye® Subclass Specific Antibodies Technical Note


Tip: Use freshly-made buffers to prevent contamination.

Detect with Odyssey® Imaging System

Tip: Use a detection system with a wide linear dynamic range, like the Odyssey® Imaging Systems.

Near-Infrared (NIR) Western Blot Detection Technical Note

Analyze with Empiria Studio® Software

Tip: Let Empiria Studio Software handle the background subtraction method.

Infographic Overview of the Concepts Behind Empiria Studio Workflows

Store or Archive Blots

Western Blot Storage Bags