![]() ![]() However, Captain Obvious would point out that this type of analysis has severe limitations and is not useful for precise signal quantification. “Eyeballing it” may be fine if you’re looking for the presence or absence of a signal, or if the signal intensities you are comparing differ significantly (unless, of course, you forget to remove your sunglasses). In other words, you hold the film up to the light, cock your head like a dog’s at the sound of the word “treat”, and strain your eyes as if trying to decipher the fine print on the back of credit card application. Using film to develop blots has traditionally been associated with a “visual assessment” of relative band intensities. Sensitivity / Dynamic Range / Limits of Detection As a disclaimer, this information was compiled over years of experience and to a lesser extent represents this author’s humble opinion. Let’s look at the individual inherent strengths and weaknesses of the two methods. So, with western blotting so prevalent in so many different labs, which is the ‘better’ option – traditional film development, or CCD imaging? That is a difficult question to answer – needs, practicality, expertise, funding and applications vary from lab to lab. With the advent of charged-coupled device (CCD) camera technology, new options for detecting, analyzing and capturing images are readily. quality of materials, proficiency of the user, strength of the protocol, etc.), the wide dynamic range and rapid results have made this the method of choice for most labs over the past 30 years. While there are a number of factors affecting the performance of chemiluminescent imaging (e.g. It’s also a much safer alternative to radioactive probes, which require you to wear enough Nomex to dance at Chernobyl. Since the late 1970’s, chemiluminescence has proven to be a reliable and highly sensitive method for detecting and quantifying proteins. Once the shrieking subsides, you may be able to locate the developer and develop your blot…but your colleague will never go back in the darkroom with you again. But now the lights are out, you can’t find your film cartridge, and, while awkwardly grasping in the darkness for your blot, you’ve managed to grope your startled colleague. It didn’t seem so imposing when the lights were on, and plus, you’ve brought a friend to help. Darkroom – ah yes, that magical place where night vision goggles are required to navigate a veritable minefield of potential chaos. You’ve got your antibodies and ECL ready to go. You’ve masterfully run and transferred your gel, and now it’s time to probe and quantify your protein(s).
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