Protocol for

Simultaneous Chemiluminescent and Fluorescent Detection

Link to Full Protocol: http://www.carestreamhealth.com/AP0106-s11-chem-fluo-detecti ...

Abstract or Description:

A novel approach for double Western blot analysis of low copy protein targets

The ability to multiplex fluorescent labels has greatly improved the utility of applications like Western blotting providing both cost and time-savings ability to detect two or more protein targets simultaneously. However, chemiluminescent Western blot analysis remains the most sensitive method for detection of low-copy protein targets. Since most loading control proteins are highly expressed proteins that can be easily detected using fluorescence, and most researchers are trying to detect low-copy proteins of interest,
we have developed a method for simultaneous incubation of two protein targets using both fluorescent and chemiluminescent secondary antibodies. The following study demonstrates
that addition of HRP substrates, prior to fluorescence imaging, has no adverse affects on the fluorescent signal of Alexa Fluor^Ò labeled antibodies. Additionally, a protocol is presented that describes both the capture of luminescent and fluorescent images, as well as post image capture processing (i.e. pseudocolor and image overlay) to produce publication quality figures.

Citation:

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Posted By: Carestream Health on 3/14/2011 11:14:06 AM

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