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Baghirova S, Hughes BG, Hendzel MJ, et al. Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells. MethodsX. 2015;2:440-5doi: 10.1016/j.mex.2015.11.001.
Baghirova, S., Hughes, B. G., Hendzel, M. J., & Schulz, R. (2015). Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells. MethodsX, 2440-5. https://doi.org/10.1016/j.mex.2015.11.001
Baghirova, Sabina, et al. "Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells." MethodsX vol. 2 (2015): 440-5. doi: https://doi.org/10.1016/j.mex.2015.11.001
Baghirova S, Hughes BG, Hendzel MJ, Schulz R. Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells. MethodsX. 2015 Nov 07;2:440-5. doi: 10.1016/j.mex.2015.11.001. eCollection 2015. PMID: 26740924; PMCID: PMC4678309.
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MethodsX. 2015 Nov 07;2:440-5. doi: 10.1016/j.mex.2015.11.001. eCollection 2015.

Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells.

MethodsX

Sabina Baghirova, Bryan G Hughes, Michael J Hendzel, Richard Schulz

Affiliations

  1. Department of Pharmacology, Cardiovascular Research Centre, Mazakowski Alberta Heart Institute, University of Alberta, Edmonton, Alberta, Canada.
  2. Department of Pharmacology, Cardiovascular Research Centre, Mazakowski Alberta Heart Institute, University of Alberta, Edmonton, Alberta, Canada; Department of Pediatrics, Cardiovascular Research Centre, Mazakowski Alberta Heart Institute, University of Alberta, Edmonton, Alberta, Canada.
  3. Department of Oncology, University of Alberta, Edmonton, Alberta, Canada.

PMID: 26740924 PMCID: PMC4678309 DOI: 10.1016/j.mex.2015.11.001

Abstract

Many types of studies require the localization of a protein to, or isolation of enriched protein from a specific cellular compartment. Many protocols in the literature and from commercially available kits claim to yield pure cellular fractions. However, in our hands, the former often do not work effectively and the latter may be prohibitively expensive if a large number of fractionations are required. Furthermore, the largely proprietary composition of reagents in commercial kits means that the user is not able to make adjustments if, for example, a particular component affects the activity of a protein of interest. The method described here allows the isolation of purified proteins from three cellular fractions: the cytosol, membrane-bound organelles, and the nucleus. It uses gentle buffers with increasing detergent strength that sequentially lyse the cell membrane, organelle membranes and finally the nuclear membrane.•Quick, simple to replicate or adjust; this method does not require expensive reagents or use of commercial kits•The protocol can be applied to tissue samples or cultured cells without changing buffer components•Yields purified fractions of cytosolic, membrane bound and nuclear proteins, with the proper distribution of the appropriate subcellular markers: GAPDH, VDAC, SERCA2 and lamin A/C.

Keywords: Cell fractionation; Membrane proteins; Nuclear proteins; Sequential lysis; Subcellular organelles; Tissue fractionation

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