Western blot: technique, theory, and trouble shooting

Abstract

Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. This paper will attempt to explain the technique and theory behind western blot, and offer some ways to troubleshoot.

Keywords: Bio-medical research; protein; western blot.

Figure 1

Assembled rack for gel solidification

Figure 2

Add gel solution using a transfer pipette

Figure 3

Add running buffer to the electrophorator

Figure 4

Add samples and molecular marker to the gel, after removing the combs

Figure 5

(a) Samples running through the stacking gel (lower voltage). (b): Samples running through the separating gel (higher voltage)

Figure 6

Run the gel to the bottom of the electrophorator

Figure 7

Transfer should be done on ice

Figure 8

Membrane after transfer

Figure 9

Use a shaker to incubate the membrane with antibody

Figure 10

Incubate the membrane with ECL mix using a 1000 μL pipette to help the process

Figure 11

Use the cassette to expose the membrane in the dark room

Figure 12

Assembly of a sandwich in western Blot