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Distortion of protein analysis in primary neuronal cultures by serum albumin from culture medium: A methodological approach to improve target protein quantification.

Journal of neuroscience methods (2018-07-24)
Ashleigh Willis, Judith A Pratt, Brian J Morris
ABSTRACT

Primary neuronal cultures underpin diverse neuroscience experiments, including various protein analysis techniques, such as Western blotting, whereby protein extraction from cultured neurons is required. During immunoblotting experiments, we encountered problems due to a highly-abundant protein of 65-70 KDa present in the cell extracts, that interfered with total protein estimation, and immunodetection of target proteins of similar size. Previous research has suggested that serum proteins, specifically albumin, contained within commonly-used culture media, can bind to, or be adsorbed by, generic cell culture plasticware. This residual albumin may then be extracted along with cell proteins. We made simple modifications to wash steps of traditional cell lysis/extraction protocols. We report that a substantial amount of albumin, accumulated from the standard culture media, is extracted from primary neuronal cultures along with the cellular contents. This contamination can be reduced, without changing the culture conditions, by modifying wash procedures. Accumulated albumin from neuronal culture media, in amounts equivalent to cellular contents, can distort data from total protein assays and from the immunoreactive signal from nearby bands on Western blots. By altering wash protocols during protein extraction, these problems can be ameliorated. We suggest that the standard extended culture periods for primary neuronal cultures, coupled with the requirement for successive medium changes, may leave them particularly susceptible to cumulative albumin contamination from the culture media used. Finally, we propose the implementation of simple alterations to wash steps in protein extraction protocols which can ameliorate this interference.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Brilliant Blue G, 250, for microscopy
Sigma-Aldrich
p-Toluenesulfonic acid monohydrate, ≥99% (calc. to H2O free subst.)
Sigma-Aldrich
Anti-Glutamic Acid Decarboxylase 65/67 antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution
Millipore
SignalBoostImmunoreaction Enhancer Kit