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Nuclear-Cytosol Extraction Kit (100 assays, 5-10 ( 106 cells per assay) #P1200 This Nuclear-Cytosol Extraction Kit provides a complete system for preparing both nuclear and cytoplasmic protein extracts from mammalian tissue or cultured cells. The kit provides flexibility for optimal protein extraction, for example, the choice between detergent and non-detergent extraction. With the optimized reagents and procedures, the extracts can be prepared in less than 1 hour and are highly pure since there is little or no cross-contamination between nuclear and cytoplasmic extracts. The extracted nuclear and cytoplasmic protein fractions can be used in downstream assays such as transcriptional activity, gel shift assay, reporter assays, enzyme activity assays, immunoprecipitation, and Western blotting. Kit Contents and Storage Cytosol Extraction Buffer A (CEB-A) 50 ml, 4 oC; Cytosol Extraction Buffer B (CEB-B) 2 x 1.5 ml, 4 oC Nuclear Extraction Buffer (NEB) 10 ml, 4 oC. The kit is sufficient for 100 extractions of each 5 ( 106 cells. The kit compositions are stable at least for 1 year at 4 oC. Note that protease inhibitors are not included in buffer. User can add protease inhibitors, although usually all buffers work fine without addition of the inhibitors. General Consideration Keep all buffers on ice and handle the sample on ice or at 4oC. Estimation of packed cell volume (PCV): PCV is an estimated volume of the pelleted cells after centrifugation. The volume of PCV is dependent on cell types. The 1 x 106 packed cells usually give a 10-20 (l PCV. The protocol is described for fractionation of up to 5~10 x 106. If less or more cells are extracted, proportionally scale up the initial volume of the extraction buffers. Cell Numbers 5 x105 1 x106 5 x106 Estimated Packed Cell Volume (PCV, (l) 5 10-20 60-100 Cytosol Extraction Buffer A ((l) 25 50-100 300-500 Cytosol Extraction Buffer B ((l) 1.2-1.5 3-6 18-30 Nuclear Extraction Buffer ((l) 5-10