title: The fastest Western in town: a contemporary twist on the classic Western blot analysis.
abstract: The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.
authors.affiliation: Diller Cancer Research Building, University of California, San Francisco; SilvaJi@cc.ucsf.edu.
authors.forename: Jillian M
authors.initials: JM
authors.lastname: Silva
authors.affiliation: Diller Cancer Research Building, University of California, San Francisco.
authors.forename: Martin
authors.initials: M
authors.lastname: McMahon
mesh_terms: D015153:Blotting, Western / Q000295:instrumentation / Q000379:methods* / Q000639:trends; D006801:Humans; D008545:Melanoma / Q000737:chemistry / Q000378:metabolism; D009363:Neoplasm Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D009363:Neoplasm Proteins; D011506:Proteins