a housekeeping gene or protein that should not vary between samples given the treatment, such as GAPDH or beta-Actin). For these reasons, X-ray film should never be used for quantitative purposes and a normalized loading control should always be present (i.e. This being said, X-ray film lacks the linearity and range of detection as a CCD. Given the low expression rates of some of the studied proteins in our lab, we have yet to see comparable sensitivity with a CCD under the same blotting conditions and exposure time. In fact, chemiluminescence coupled with X-ray film is the most sensitive method for western blot detection. Between the enzymatic methods of western blot detection, chemiluminescence is far more sensitive and more commonly used than colorimetric methods. Light intensity can then be measured by exposure to an X-ray film or a charged coupled device (CCD) that converts photons to an electronic signal. Detection method: CCDĪdvantages: Easy to use and more linear than film.ĭisadvantages: Possibly less sensitive than film, cost of a CCD, single output signal.Ĭhemiluminescence works by producing light as a byproduct from a reaction between a chemical substrate and the enzyme. Chemiluminescence Detection method: FilmĪdvantages: High sensitivity (femtogram range).ĭisadvantages: Non-linear, small range of detection, single output signal, requires dark room, continuous cost of film and developer maintenance. ![]() Given the low sensitivity of colorimetric methods, I would only recommend these techniques for non-quantitative purposes with highly expressed proteins. Though I believe it may still be used for undergraduate laboratory classes. I personally have never witnessed any current research labs that still use colorimetric detection methods for western blotting (I’m sure there’s probably a few out there still). However, their range of sensitivity requires 5-500 pg of protein much higher than chemiluminescent sensitivity, which can be in the femtogram range. Colorimetric assays are generally easier to use and don’t require the costly materials of X-ray film or a developer. For example, HRP catalyzes a reaction with 4-Chloro-1-napthol (4CN) and peroxide that produces a visible and insoluble purple product. It does not store any personal data.Advantages: Cheap, easy to use, quick results.ĭisadvantages: Low sensitivity (picogram range), only single output signal.Ĭolorimetric assays act by producing a colored precipitate from an enzymatic reaction. The cookie is set by the GDPR Cookie Consent plugin and is used to store whether or not user has consented to the use of cookies. The cookie is used to store the user consent for the cookies in the category "Performance". This cookie is set by GDPR Cookie Consent plugin. The cookies is used to store the user consent for the cookies in the category "Necessary". ![]() ![]() The cookie is used to store the user consent for the cookies in the category "Other. The cookie is set by GDPR cookie consent to record the user consent for the cookies in the category "Functional". The cookie is used to store the user consent for the cookies in the category "Analytics". These cookies ensure basic functionalities and security features of the website, anonymously. Necessary cookies are absolutely essential for the website to function properly. Protein and Western Blotting Reagents and Accessories.
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