Blue or Gray – Is Color Important for Western Blot Film?

Western blot film – some is blue some is gray.  Some is even clear.  Then there are the boxes of pre-exposed film with black edges and corners that you only use when you have no other film left.

Does it make a difference which film you use for your experiment (other than the pre-exposed film, that is)?

A little bit of knowledge will help you decide.  Let’s look at the differences between film and the important things to consider when choosing film for your experiment:

How films are similar

Most x-ray film has 4 properties in common:

1. It is made of a polyester base that supports the light-sensitive emulsion.  (And you thought polyester was so 70’s.)
2. It has an outer layer that protects the film from scratches, static, etc.  But it is not perfect, so be nice to your film.
3. It is coated on both sides with emulsion (watch out for the special kinds that aren’t!).  This is good for you as you don’t have to figure out in the dark room which side of the film to expose to your blot.  It also increases the film speed and resolution, but could cause blurring.
4. It is sensitive to blue and green, but not red light.  So you can leave those red lights on in the dark room!

How films are different

Speed and resolution

Speed and resolution, and not color,  are actually the two most important criteria for choosing your film.  They are determined by the size of the silver halide crystals and the thickness of the emulsion.

• Large crystals and thick emulsions = increased sensitivity but decreased resolution
• Smaller crystals and thinner emulsions = high resolution and decreased sensitivity

If you are using low energy radioisotopes like ³³P or ¹⁴C, then use high resolution film.  For chemiluminescence, use a high-performance film like LucentBlue.  Or, be on the safe side and buy an all-purpose film like Kodak BioMax, which has high sensitivity without compromising resolution.

Color – blue or gray…or clear?

As long as your film has the right resolution and sensitivity for your application, then color isn’t as important.  However, blue film is believed to reduce glare and eyestrain, which may help you out if you spend a lot of time looking for bands.  Some people also find that their bands appear to “stand out” better on a blue background versus gray.  Watch out though, blue film can be less sensitive and might not be the right choice for low abundance proteins.

And then there are clear films that turn completely transparent (except for your bands) when developed.  These might help you see very faint bands.

Price

The price of Western blot film can vary greatly.  A sheet of film can cost anywhere from $0.50-$10! Film that is individually wrapped is more expensive, while blue film tends to cost less.

Which Western blot film is right for you? 

Different experiments may require different film.  Most Western blots are easily visualized on economical blue Western blot film. You can use this less expensive film for general use and troubleshooting, or if you know you are going to need to do a lot of exposures.  Save the expensive film for hard-to-detect bands and for final experiments.

Your boss will thank you.

Photo courtesy of www.GlynLowe.com.