Enrichment of Cytoskeleton Proteins

  • Fig. 1: Scheme of the cytoskeleton enrichment procedureFig. 1: Scheme of the cytoskeleton enrichment procedure
  • Fig. 1: Scheme of the cytoskeleton enrichment procedure
  • Fig. 2: Western blot of compartmental proteins extracted from HeLa cells. Results indicate that GAPDH (Panel A) is present in the soluble cytoplasmic (lane S) and nuclear (lane N) fractions, and the intermediate filament protein Vimentin (Panel B) is present exclusively in the cytoskeletal compartment (lane C).
  • Fig. 3: Non-treated/non-enriched and treated/enriched HeLa cells. (A) F-actin was detected using TRITC-conjugated phalloidin, (B) cytosolic protein was detected using GAPDH antibody as a cytosolic control and a FITC-conjugated secondary, (C) nuclear counterstaining was revealed with DAPI and all images were overlaid. Soluble cytosolic protein fraction (GAPDH) was successfully removed after enrichment.
  • Fig. 4: Non-treated/non-enriched and treated/enriched HeLa cells. (A) F-actin was detected using TRITC-conjugated phalloidin, (B) focal adhesion contacts were detected using vinculin antibody and an FITC-conjugated secondary, (C) nuclear counterstaining was revealed with DAPI and all images were overlaid. Background due to soluble cytosolic fraction was significantly reduced after enrichment, resulting in clear detection of insoluble, actin-associated proteins (vinculin).
  • Fig. 5: Non-treated/non-enriched and treated/enriched HeLa cells. (A) F-actin was detected using TRITC-conjugated phalloidin, (B) cell junctions were detected using b-catenin antibody and a FITC-conjugated secondary, (C) nuclear counterstaining was revealed with DAPI and all images were overlaid. Background due to soluble cytosolic fraction was significantly reduced after enrichment, resulting in clear detection of insoluble, low-abundance actin-associated proteins (b-catenin).

The actin cytoskeleton is comprised of actin polymers and associated proteins. It mediates a variety of biological functions in eukaryotic cells. Studying proteins that associate with and regulate the actin cytoskeleton has been difficult because of the insolubility of the cytoskeleton in traditional detergents. Here we describe a cytoskeleton purification and enrichment method that allows for rapid and convenient selective enrichment of proteins associated with the cytoskeleton for biochemical and cell visualization analyses.

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