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.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
For adherent cells
For both adherent and suspension cells
Table 1: Volumes in μl for WCL or buffer at indicated cell numbers.
|2.5 x 106 cells||5 x 106 cells||7.5 x 106 cells||1 x 107 cells|
Protocol Id: 584
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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