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Methods Mol Biol. 2015;1295:381-91. doi: 10.1007/978-1-4939-2550-6_27.

Western blotting using in-gel protein labeling as a normalization control: stain-free technology.

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Department of Neurobiology, Physiology, and Behavior, University of California, Davis, 191 Briggs Hall, One Shields Avenue, Davis, CA, 95616, USA.


Western blotting is a commonly used laboratory technique for semi-quantifying protein amounts. It is important when quantifying protein expression to account for differences in the amount of total protein loaded onto the gel using a loading control. Common loading controls include housekeeping proteins, such as β-actin or GAPDH, quantified by Western blot, or total protein, quantified using a stain such as Coomassie Brilliant Blue or Ponceau S. A more recently developed method for total protein quantification utilizes stain-free technology, which has a linear dynamic detection range and allows for protein detection on both gels and membranes. Here, we describe the theory and use of stain-free gels for total protein quantification and normalization of Western blots.

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