Western blot analysis of cell fractions from HeLa cells using MEK1/2 (D1A5) Rabbit mAb #8727, AIF (D39D2) XP® Rabbit mAb #5318, Histone H3 (D1H2) XP® Rabbit mAb #4499, and Vimentin (D21H3) XP® Rabbit mAb #5741 (as part of the Cell Fractionation Antibody Sampler Kit #11843) showing cytoplasmic, organellular/membrane, and nuclear/cytoskeletal localization. Whole cell lysates (WCL) were used to represent total protein. Cytoplasmic proteins (Cyto) were isolated using CIB buffer. Integral membrane and organellular proteins (Mem) were isolated using MIB buffer. Nuclear and cytoskeletal proteins (Nuc) were isolated using CyNIB buffer.
|Cytoplasmic Isolation Buffer (CIB)||10 ml|
|Membrane Isolation Buffer (MIB)||10 ml|
|Cytoskeletal/Nuclear Isolation Buffer (CyNIB)||5 ml|
|Protease Inhibitor Cocktail (100X) 5871||250 µl|
Store at -20°C. Upon receipt, Protease Inhibitor Cocktail 5871 should be stored at 4°C. All other components should be stored at -20°C.
The Cell Fractionation Kit is designed to provide a fast and efficient way of separating cultured cells into three distinct fractions: cytoplasmic, membrane/organelle, and nuclear/cytoskeletal. These fractions can then be analyzed by SDS-PAGE and western blotting. The kit includes enough buffer for 20 assays.
Cellular fractionation allows for the extraction of cellular proteins into distinct compartments. This is achieved by the use of detergents that take advantage of the inherent qualities and composition of different cellular membranes (1). Cellular fractionation has been important for defining the localization of many proteins, observing the translocation of proteins, and determining protein-protein complexes, such as cytoskeletal-associated proteins (2,3). Thus, detergent-based cellular fractionation separates cellular components with greater ease and speed compared to a more laborious density centrifugation method (4).
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