Western Blotting

Western Blot visual protocol: phase 1: Sample Preparation

In phase 1 of the western blot procedure, you will learn how to prepare your samples before loading them into a gel. Here we isolate protein from cultured cells, quantify total protein concentrations with a BCA assay, add loading buffer to the sample, and heat the sample.

Western Blot visual protocol: Phase 2: Protein electrophoresis (SDS-PAGE)

In phase 2 of the western blot procedure, you will learn how to load a gel and separate the proteins through electrophoresis, based upon protein weight.

Demystifying SDS-PAGE

Western Blot visual protocol: phase 3: Membrane transfer

In phase 3 of the western blot procedure, you will learn how to transfer your proteins from the SDS-PAGE gel onto a PVDF or nitrocellulose membrane. Here we remove the gel from the cassette, and stack it in a sandwich comprised of a sponge, filter paper, the gel, membrane, filter paper, and sponge. The negatively charged proteins will then transfer onto the membrane toward the positive current.

Western Blot visual protocol: phase 4: Immunoblotting

In phase 4 of the western blot procedure, you will learn how to block the membrane, stain in with Ponceau red, and add the primary and secondary antibodies. Vigorous washing when indicated between these steps is essential for obtaining a clean blot.

Western Blot visual protocol: phase 5: Detection

In phase 5 of the western blot procedure, you will learn how to visualize your protein of interest that was probed with specific antibodies in the previous step. Here we utilize the electrochemiluminescent (ECL) to produce light where our antibodies are bound. This light is collected by film or a camera for later analysis.