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

Ashleigh Willis, Judith A. Pratt, Brian J. Morris

Research output: Contribution to journalArticle

Abstract

BACKGROUND: 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.

NEW METHOD: We made simple modifications to wash steps of traditional cell lysis/extraction protocols.

RESULTS: 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.

COMPARISON WITH EXISTING METHODS: 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.

CONCLUSIONS: 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.

LanguageEnglish
Pages1-5
Number of pages5
JournalJournal of Neuroscience Methods
Volume308
Early online date6 Jul 2018
DOIs
Publication statusPublished - 1 Oct 2018

Fingerprint

Serum Albumin
Culture Media
Albumins
Proteins
Western Blotting
Neurosciences
Cell Extracts
Immunoblotting
Blood Proteins
Cell Culture Techniques
Neurons
Research

Keywords

  • band distortion
  • protein adsorption
  • immunoglot problem
  • protein extraction
  • primary neurons

Cite this

@article{cb8c83f52ca34541a923b8c82700e8c0,
title = "Distortion of protein analysis in primary neuronal cultures by serum albumin from culture medium: a methodological approach to improve target protein quantification",
abstract = "BACKGROUND: 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.NEW METHOD: We made simple modifications to wash steps of traditional cell lysis/extraction protocols.RESULTS: 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.COMPARISON WITH EXISTING METHODS: 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.CONCLUSIONS: 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.",
keywords = "band distortion, protein adsorption, immunoglot problem, protein extraction, primary neurons",
author = "Ashleigh Willis and Pratt, {Judith A.} and Morris, {Brian J.}",
note = "Copyright {\circledC} 2018 Elsevier B.V. All rights reserved.",
year = "2018",
month = "10",
day = "1",
doi = "10.1016/j.jneumeth.2018.07.002",
language = "English",
volume = "308",
pages = "1--5",
journal = "Journal of Neuroscience Methods",
issn = "0165-0270",

}

TY - JOUR

T1 - Distortion of protein analysis in primary neuronal cultures by serum albumin from culture medium

T2 - Journal of Neuroscience Methods

AU - Willis, Ashleigh

AU - Pratt, Judith A.

AU - Morris, Brian J.

N1 - Copyright © 2018 Elsevier B.V. All rights reserved.

PY - 2018/10/1

Y1 - 2018/10/1

N2 - BACKGROUND: 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.NEW METHOD: We made simple modifications to wash steps of traditional cell lysis/extraction protocols.RESULTS: 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.COMPARISON WITH EXISTING METHODS: 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.CONCLUSIONS: 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.

AB - BACKGROUND: 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.NEW METHOD: We made simple modifications to wash steps of traditional cell lysis/extraction protocols.RESULTS: 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.COMPARISON WITH EXISTING METHODS: 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.CONCLUSIONS: 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.

KW - band distortion

KW - protein adsorption

KW - immunoglot problem

KW - protein extraction

KW - primary neurons

U2 - 10.1016/j.jneumeth.2018.07.002

DO - 10.1016/j.jneumeth.2018.07.002

M3 - Article

VL - 308

SP - 1

EP - 5

JO - Journal of Neuroscience Methods

JF - Journal of Neuroscience Methods

SN - 0165-0270

ER -