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Evaluation of rapid western blot incubation system
Ist Teil von
The FASEB journal, 2010-04, Vol.24 (S1), p.900.2-900.2
Ort / Verlag
Federation of American Societies for Experimental Biology
Erscheinungsjahr
2010
Link zum Volltext
Quelle
Wiley Online Library
Beschreibungen/Notizen
Western Blot has been a powerful tool in life science labs for over thirty years. One of the encumbrances of this method is that it is labor and time intensive. In our lab, shortened incubation times are acceptable for identity testing, however long incubation times are still standard when detecting low amounts of antigen or when using low affinity antibodies. We evaluated the Millipore SNAP i.d™ for use in routine western blot analysis as well as for researching new proteins whose properties were not as well known. The SNAP i.d.™ uses vacuum assisted diffusion to greatly decrease incubation times. A variety of samples including recombinant proteins and antibody fragments were optimized for both methods. Proteins were transferred using the Invitrogen i‐Blot®. Direct dual‐development analysis, staining the blot with Sypro® Ruby prior to antibody exposure was used to differentiate closely migrating proteins from the protein of interest. Commercially available kits were utilized to develop the blots. This study indicated that the SNAP i.d.™ is suitable for a variety of applications of the Western blot technique, although substantial method development may be required for unfamiliar proteins and specific applications. The system was useful for quickly identifying epitopes in specific recombinant degradation products, aiding in the identification of the specific cleavage sites and detecting low level contaminants.