Several common laboratory techniques, including Northern, Southern and Western blotting, require the capture of images generated by either P32-labeled probes or chemiluminesence substrates. While many laboratories have moved to digital imaging systems for capturing these images, X-ray film is still widely used for these applications. Film can still provide several advantages over digital systems. In many cases film and developers are readily available; film also provides higher resolution than all but the best and most expensive digital systems. However, film can provide more of a challenge when attempting to optimize exposure and development times to generate that perfect image for publication. Overexposed film can often be difficult to interpret as it is difficult to precisely determine band localization and relative intensities. Poor blocking or high concentrations of reagents can cause shading and/or spots on the film. In some cases, the length of exposure and concentration of reagents necessary to produce appropriately specific staining leads to non-specific staining which then prevents visualization of the specific pattern.
Thermo Scientific claims that all of these problems can be alleviated through application of their Erase-It Background Eliminator Kit. Their claim is that this product will reveal valuable data hidden in previously unusable blots and that unsightly spots and shading can be removed without loss of specific staining, resulting in publication quality images from draft quality blot films. So is their claim accurate? In our hands, the answer is, sometimes, to a limited extent.
The kit consists of two bottles of solution which are sufficient to make approximately three liters of working solution. The protocol recommends making 270 ml of solution to “erase” a single 5”x7” blot. I have found that most small blots can be stripped using about one tenth of the recommended volume, thus allowing you to clean ten-fold more blots than their protocol would suggest. If protected from light, the solution can be used multiple times over the course of several hours with success. The solution starts out a viscous yellow and loses color as it degrades, which provides a helpful indicator as to its useability.
When using the eliminator, you must exercise caution. Initially changes appear to be quite slow, but as more staining is removed the reaction speeds. I recommend stopping the de-staining often to document your results as it is very easy to lose your specific staining right along with the background if you should let the reaction proceed too long. Basically, the product works by removing the deposited silver grains present in the film. This means that you are removing all staining, not just that which is non-specific. In essence you are creating shorter exposures of your blots in reverse.
While this product is not the magic remedy for failed blots the product insert would have you believe, it is still a useful tool which will allow you to recover data from blots that were otherwise unusable. We use it on occasion to help remove spotting present on films or to lower background in cases where we just cannot determine the optimum exposure. Two points of caution, however, letting the reaction proceed too far will result in loss of all staining on the blot and the bright yellow solution will sometimes stain your films a yellowish tint. Overall, I find that Erase-It is a useful reagent to have on hand, but do not count on it to replace optimization in your experimental life.