Uploaded January 2022 | Updated September 2026, 2 weeks ago

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Watch this video to explore considerations for optimizing incubation time and dilution of primary antibodies when performing a western blot.

Commercially purchased antibodies should have a starting dilution recommended by the manufacturer. Plan your dilution series around the manufacturer-recommended dilution. This should help establish the optimal dilution for your specific target protein.

Once the optimal primary antibody dilution has been established, you can optimize incubation time. The typical recommendation for primary antibody incubation is 1 to 2 hours at room temperature or overnight at 4 degrees Celsius. The optimal choice depends on your antibody and sample.

However, a longer incubation time may result in higher background and non-specific binding. These issues should be minimal if the primary antibody dilution has already been optimized.

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