July 10, 2017

Why choose phosphorimaging for Western blots?

By Cytiva

Read about the benefits of phosphor screens over x-ray film in high-sensitivity Western blot imaging.

Autoradiography for high-sensitivity Western blotting

Two methods of autoradiography are used in Western blotting:
  1. X-ray film autoradiography
  2. Storage phosphor screen autoradiography (based on photostimulated luminescence)

Scientists have been using X-ray filmautoradiography since radioactive isotopes were first used to tag biomolecules. Films remaina detection option in today’s laboratories but new imaging techniques are quicklytaking over.Autoradiography on photographic film converts emitted radiation into light. This conversion helps to increase the otherwise limited sensitivity of the radioisotope. However, this gain in sensitivity coupleswith decreased resolution and non-linear responses.

Alternatively, phosphorimaging offers a highly sensitive optionfor imagingWestern blots. The method deploys storage phosphor screens, which are up to 100-fold more sensitive than film for radioisotopes.

How does a phosphor screen work?

Each phosphor screen comprises a three-layer phosphor imaging plate. A photostimulable phosphor layer is sandwiched between a protective layer and a support film.

When exposed to high-energy radiation, photostimulatable phosphor crystals excite and trap electrons. Placing the screen in an imager/laser scanner exposes it to visible light, releasing electrons to return to their ground state. An imager detects the emitting photons, producinga digital image that shows the location of radioisotopes.

In Western blot imaging, bands appear on the digital image wherever proteins labeled with radioisotopes are located.

As well as producing a visual representation of the blot, laser scanners (such as Typhoon) can precisely quantitate the radiolabeled proteins. This is because the amount of radioactivity is directly proportional to the amount of labeled protein.

Film vs phosphor screen

Storage phosphor screens overcome many of the issues associated with using film for radioactive detection:

1. Time

The exposure time for a film screen is significantly longer than for a phosphor screen. Exposure time for X-ray film is typically between 24 and 72 h. Phosphorimaging can be performed in 3 h or lessdue to increased detector efficiency.

2. Sensitivity

The high sensitivity of phosphor imaging is 10 to 100-fold that of film screens. This large range is dependent on the isotope used and is the result of high-density excitable particles on the phosphorimaging storage plate.

3. Dynamic range

The dynamic range for phosphor imaging (five orders of magnitude) is broader than for film (two orders of magnitude).A wider dynamic range is beneficial in imaging as it enables capturing of a wider range of signal intensities in the same exposure.

4. Logistics

Phosphorimaging does not require development chemicals or a dark room, which are both necessary for film. Storage phosphor screens also work on a variety of media including the gels themselves, membranes, microarray plates, and tissue slices.

5. Reusability

Storage phosphor screensare reusable as they can be erased following each use. In comparison, film creates a permanent record. Combined with the lack of chemicals required, phosphorimaging helps to reduce waste in the lab.