Removes overexposure speckling, haze and background from developed X-ray film.
Thermo Scientific Pierce Background Eliminator provides fast, easy removal of haze and artifacts on developed X-ray film to correct for overexposure of blots, whether from chemiluminescent Western blot or DNA or RNA autoradiography.
This two-reagent kit is simple to use as demonstrated in the video below. Mix the two reagents (1:1 ratio) and immerse a developed piece of film (recent or old) in the working solution. Watch for 1 to 5 minutes until the desired image appears, then stop the reaction by rinsing the film in water. Pierce Background Eliminator for Film evenly reverses development-signal over the entire film, preserving relative densitometry values so that analysis and publication of results is still valid and possible.
For overexposed X-ray film – reverses development of silver-based autoradiography film removing silver emulsion from the film surface
Recovers and preserved data – evenly reduces developed signal over the entire film so relative intensity (densitometry) of bands is not altered
Optimizes for signal-to-noise – allows readjustment of overdeveloped film to obtain the best image with respect to background haze, speckles and oversaturated bands that need to be resolved
Saves time and resources – eliminates the need to repeat experiments or re-expose a new piece of film to obtain an optimal image
Video of Thermo Scientific Pierce Background Eliminator for Film in action!
X-ray film developed from enhanced chemiluminescence Western blots of any kind, whether from Thermo Scientific SuperSignal, Pierce ECL or other commercial substrate
X-ray film developed from any chemiluminescent or radioactive gel-shift assays, including Thermo Scientific LightShift Chemiluminescent EMSA Kits for DNA or RNA
Autoradiography film developed from chemiluminescent or isotopic (32P or 35S) Northern or Southern blots, including with Thermo Scientific North2South Chemiluminescent Detection Kit
Any developed silver-emulsion photographic film, whether blue, clear, single- or double-sided, and regardless of the method of exposure
High background, shading, overexposed bands and speckling are problems inherent to film exposure. High background and shading are caused by overexposure, poor use of blocking buffer or inappropriate enzyme-labeled probe or antibody concentration. Overexposed bands are a common occurrence when the enzyme-labeled probe or antibody used is too concentrated or if the film was exposed for too long. Speckling and shading occur when enzyme conjugates form complexes and precipitate on the blot. Pierce Background Eliminator can correct all these problems without the need to re-expose a blot to film or re-do an entire experiment. The working solution can be used with newly exposed films or those that have been stored for years. In addition, the product can be used with any brand of film.
Thermo Scientific Pierce Background Eliminator recovers overexposed and overdeveloped film in less than five minutes. A431 cell lysate was electrophoresed on a 4-12% Tris-glycine gel and transferred overnight to nitrocellulose. The membrane was blocked with Thermo Scientific SuperBlock Blocking Buffer in PBS (Part No. 37515) for 1 hour and incubated with 1.25ng/mL of HRP-labeled mouse anti-phosphotyrosine (PY20) for 1 hour. After the membrane was washed for 30 minutes, Thermo Scientific SuperSignal West Dura Substrate (Part No. 34075) was added. The blot was exposed to film for 10 seconds and resulted in a completely black image caused by the antibody cross-reacting with the blocking buffer (A). Using the old option (B), another gel and the entire procedure was repeated, this time using 5% dry milk solution for blocking and 2.5ng/mL PY20 antibody. Using the new option (C), the original dark film (A) was treated with Pierce Background Eliminator to allow the band images to appear in 4 minutes.
Thermo Scientific Pierce Background Eliminator lightens and resolves overexposed bands. Recombinant human wild-type p53 baculovirus lysate was separated on a 12% SDS-polyacrylamide gel. The proteins were transferred to a nitrocellulose membrane and blocked with SuperBlock Blocking Buffer in PBS (Part No. 37515). The protein was detected with mouse anti-p53 followed by goat anti-mouse-HRP (Part No. 31434) and SuperSignal West Pico Substrate (Part No. 34080). The membrane was exposed to film for 1 minute (A). The film had overexposed bands and was treated with Pierce Background Eliminator for 6 minutes. The resulting image provided better visualization of the different p53 protein bands (B).
Densitometry data on dot blot comparing before and after use of the Thermo Scientific Pierce Background Eliminator. Dot blots were prepared on nitrocellulose (Part No. 77010) using Biotinylated-BSA (Part No. 29130) at 1000, 250, 62.5 and 15.6pg. The blot was blocked with SuperBlock Blocking Buffer in PBS (Part No. 37515) and incubated with a 1/50,000 dilution of Streptavidin-HRP (Part No. 21126). The blot was then washed for 30 minutes, incubated in SuperSignal West Pico Substrate (Part No. 34080) and exposed to film (Part No. 34092) for 5 minutes. The resulting film had high background that was cut into four strips each containing three replicates per concentration. The Background Eliminator working solution was used on separate film strips at 1, 2.5 and 4 minutes, leaving a control strip for comparison. After scanning on a densitometer, the relative signal intensity was compared. The signal intensity decreased evenly with time when treated with the Background Eliminator, maintaining similar slopes on a dose response curve.
Thermo Scientific Pierce Background Eliminator erases speckling. Recombinant Human TNFα was electrophoresed on a 4-20% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was blocked and detected with Mouse anti-Human TNFα followed by goat anti-mouse-HRP (Part No. 31434) and SuperSignal West Dura Substrate (Part No. 34075). The blot was exposed to film for 30 seconds, resulting in considerable background speckling (A). The film was then treated with Pierce Background Eliminator for 2 minutes to eliminate the background speckling (B).