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Optimization of stripping/reprobing for Li‐COR Western blot analysis
Author(s) -
McCauley Michael C,
Nalbandian Ari,
Brum Gabby,
Lu Shan,
Wan Yinsheng
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.703.2
Subject(s) - stripping (fiber) , membrane , reagent , chemistry , chromatography , analytical chemistry (journal) , western blot , materials science , biochemistry , composite material , gene
The Li‐Cor Odyssey Infrared Imaging System is pioneering the way for standard Western blot analysis by using direct infrared fluorescence detection. An advantage of the Li‐COR Odyssey Infrared Imaging System is that it provides a safer and cleaner method to detect proteins compared to traditional darkroom scans because each membrane does not have to be soaked in toxic detection reagents before the scan is initiated. However, one key problem with the Odyssey imaging system is that its standard stripping protocol fails to remove all of the antibodies from each membrane. In order to completely strip each membrane, some elements of the standard protocol had to be altered. We chose to manipulate the amount of time the membranes were exposed to the Newblot Stripping Buffer, the temperature at which the membranes were soaked in the buffer solution, and the concentration of the stripping buffer. We concluded that the optimal stripping procedure involved soaking the membranes in a 1:5 diluted buffer solution at 50°C for 30 minutes. Removing antibodies from a membrane is critical and our data suggests that these modifications to the original protocol offer a more effective stripping procedure.