20.109(F19):Complete antibody staining for Western blot analysis (Day4)

Introduction

Traditionally, only one species of antibody could be used on a Western blot because the detection relied on the emission of light that was collected by x-ray film. In the traditional systems the output looks like black bands on a blue or clear background. However, more recent conjugate chemistry has allowed secondary antibodies to be coupled to fluorescent tags. Today we will use infrared (IR) secondary antibodies to detect our α-FKBP12 and α-tubulin antibodies and then scan the Western blots using a specially constructed microscope located in the Lauffenburger lab to determine the level of BRCA2 in our cell lines.

Licor detection of IR-dye conjugated antibodies. Image was modified from the Odyssey manual.

The Licor Odyssey scanner consists of an inverted microscope with two lasers that excite dyes which emit light in the IR range. As depicted in the image on the right, an excitation point is created when beams from the 700 nm and 800 nm lasers (A) are focused on the scanning surface. The microscope objective (B) is focused on the excitation point and collects light from the fluorescing IR dyes. This light is passed through a dichroic mirror (C) that separates the light into two distinct signals that travel through two independent optical paths that are focused on separate silicon photodiodes (D) and detected. In the image, the first channel (E) and second channel (F) are shown separately and merged (G).

We will detect our IR-dye conjugated secondary antibodies at wavelengths of 700 and 800 nm. The 700 nm channel will appear red and the 800 nm channel will appear green. Infrared secondary antibodies provide a more flexible detection platform than the traditional Western blot detection methods that rely on colorimetric or chemiluminescent substrates. Unlike the colorimetric or chemiluminescent detection methods, IR dyes do not require a chemical reaction to occur in order for signal to be detected. This means that the output signal increases with time as the colorimetric or chemiluminescent substrate reaction proceeds -- making timing an important variable in traditional Western blot development. We remove that variable from the equation and control when we want to visualize our Western blot simply by controlling the excitation of the dye.

Protocols

Part 1: Apply primary antibody

Part 2: Apply secondary antibody

Part 3: Discuss research proposals with peers

Reagents list

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