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Fig. 4. Antibody recognizes sea urchin egg cytosol NSF and is inhibited by the immunizing peptide. (A) Affinity-purified anti-NSF antibody was tested against sea urchin egg cytosol by western blotting. 5, 10 or 15 µg of cytosol was subjected to electrophoresis as indicated. Following transfer onto PVDF, the blot was incubated with anti-NSF at a concentration of 1:2000 (~0.5 µg/ml) overnight at 4°C. Bound antibody was visualized using alkaline phosphatase-linked second antibody, enhanced chemifluorescence and Storm system fluorescence imaging. The position of putative NSF is indicated with the arrow. (B) 10 µg of cytosol was subjected to electrophoresis and western blot analysis as above but in the presence and absence of the immunizing peptide at a ratio of 3:1 (peptide:IgG). The NSF antibody was incubated on ice for 1 hour with the immunizing peptide prior to incubation with the blot. The labeled arrow indicates the position of the NSF band.





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