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Refinement of Protein Extraction Protocols for Human Peripheral Nerve Tissue

Frostadottir, Drifa LU ; Welinder, Charlotte LU ; Perez, Raquel LU orcid and Dahlin, Lars B. LU orcid (2025) In ACS Omega 10(5). p.5111-5118
Abstract

Our aim was to establish an effective method for protein extraction from freshly frozen human peripheral nerves, determine the minimum amount required for consistent protein extraction outcomes, and assess which method produced the highest number of protein identities. Five extraction methods were compared using 8 M urea and Ripa buffer using either the Bullet Blender or Bioruptor. Out of the total 2619 identified proteins, protein extraction using the Ripa buffer combined with either Bioruptor or Bullet Blender resulted in the identification of 1582 (60%) and 1615 (62%) proteins, respectively. In contrast, using 8 M urea and Bioruptor for protein extraction resulted in 1022 proteins (39%), whereas employing Bullet Blender yielded 1446... (More)

Our aim was to establish an effective method for protein extraction from freshly frozen human peripheral nerves, determine the minimum amount required for consistent protein extraction outcomes, and assess which method produced the highest number of protein identities. Five extraction methods were compared using 8 M urea and Ripa buffer using either the Bullet Blender or Bioruptor. Out of the total 2619 identified proteins, protein extraction using the Ripa buffer combined with either Bioruptor or Bullet Blender resulted in the identification of 1582 (60%) and 1615 (62%) proteins, respectively. In contrast, using 8 M urea and Bioruptor for protein extraction resulted in 1022 proteins (39%), whereas employing Bullet Blender yielded 1446 proteins (55%). Sample amounts, ranging from 0.6 to 10 mg, were prepared with consistent protein extraction outcome obtained for samples ≥1.2 mg. Combining Ripa and 8 M urea with Bullet Blender increased protein identification to 2126 (81%). Proteins were classified by their cell components, molecular functions, and biological processes. Furthermore, a subclassification of proteins involved in the extracellular matrix (ECM) was introduced. We recommend the use of Ripa buffer, in combination with 8 M urea and Bullet Blender for extracting proteins from fresh-frozen human nerves weighing ≥1.2 mg.

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author
; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
ACS Omega
volume
10
issue
5
pages
8 pages
publisher
The American Chemical Society (ACS)
external identifiers
  • scopus:85216858850
  • pmid:39959086
ISSN
2470-1343
DOI
10.1021/acsomega.4c11373
language
English
LU publication?
yes
id
7283dfa8-7aaf-4bc5-80bb-37848768ce71
date added to LUP
2025-03-24 13:37:23
date last changed
2025-06-02 16:35:47
@article{7283dfa8-7aaf-4bc5-80bb-37848768ce71,
  abstract     = {{<p>Our aim was to establish an effective method for protein extraction from freshly frozen human peripheral nerves, determine the minimum amount required for consistent protein extraction outcomes, and assess which method produced the highest number of protein identities. Five extraction methods were compared using 8 M urea and Ripa buffer using either the Bullet Blender or Bioruptor. Out of the total 2619 identified proteins, protein extraction using the Ripa buffer combined with either Bioruptor or Bullet Blender resulted in the identification of 1582 (60%) and 1615 (62%) proteins, respectively. In contrast, using 8 M urea and Bioruptor for protein extraction resulted in 1022 proteins (39%), whereas employing Bullet Blender yielded 1446 proteins (55%). Sample amounts, ranging from 0.6 to 10 mg, were prepared with consistent protein extraction outcome obtained for samples ≥1.2 mg. Combining Ripa and 8 M urea with Bullet Blender increased protein identification to 2126 (81%). Proteins were classified by their cell components, molecular functions, and biological processes. Furthermore, a subclassification of proteins involved in the extracellular matrix (ECM) was introduced. We recommend the use of Ripa buffer, in combination with 8 M urea and Bullet Blender for extracting proteins from fresh-frozen human nerves weighing ≥1.2 mg.</p>}},
  author       = {{Frostadottir, Drifa and Welinder, Charlotte and Perez, Raquel and Dahlin, Lars B.}},
  issn         = {{2470-1343}},
  language     = {{eng}},
  number       = {{5}},
  pages        = {{5111--5118}},
  publisher    = {{The American Chemical Society (ACS)}},
  series       = {{ACS Omega}},
  title        = {{Refinement of Protein Extraction Protocols for Human Peripheral Nerve Tissue}},
  url          = {{http://dx.doi.org/10.1021/acsomega.4c11373}},
  doi          = {{10.1021/acsomega.4c11373}},
  volume       = {{10}},
  year         = {{2025}},
}