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Am. J. Biomed. Sci. 2015, 7(1), 15-25; doi: 10.5099/aj150100015
Received: 1 December 2014; | Revised: 6 January 2015; | Accepted: 8 March 2015

 

A Strategic Approach for the Visualisation of the Low Abundance Protein Fatty Acid Binding Protein 7

 

S.J.R. Dean1*+, C.M. Perks2*, A. Rhodes1,3, J.M.P. Holly2, C. Jarrett2

1 Centre for Research in Biosciences, University of the West of England, Bristol

2 Learning & Research Building, School of Clinical Sciences, Southmead Hospital, University of Bristol, Bristol, BS10 5NB

3 Department of Pathology, University of Malaya Medical Centre, Kuala Lumpur

*Joint first authors

+Corresponding Author

Dr Sarah Dean

Senior Lecturer in Biomedical and Healthcare Sciences

Department of Biological, Biomedical and Analytical Science

University of the West of England

E-mail:  Sarah4.dean@uwe.ac.uk

 

Abstract

Fatty acid binding protein 7 (FABP7) is a 15kDa protein that plays a role in fatty acid transport, solubilisation and metabolism. It has been found to be overexpressed in triple negative/basal like breast cancer patients. The role of FABP7 in these breast cancers is not fully understood; in order to better understand how it may be involved with breast cancer prognosis cell line models are needed for mechanistic studies. However using a standard western blotting protocol FABP7 was not detectable in a selection of breast cancer cell lines and mRNA was in low abundance. This paper outlines the approach of modifying a western blot protocol and presents an optimised western blot protocol for the detection of FABP7 in breast cancer cell lines. The main areas considered during optimisation were, titration of primary and secondary antibodies, choice of protein lysis buffer, lysate preparation and a comparison between in-house 12% polyacrylamide gels and commercially available gradient polyacrylamide gels. This strategy could be used for other low abundant and small proteins.

Keywords: FABP7, western-blotting, breast cancer, protein, lysis.

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