Home Biochemistry A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample
Biochemistry JoVE (Open Access) Citable · DOI

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

DOI: 10.3791/55802-v
What you'll learn
  • Perform tissue grinding and aliquoting
  • Execute phase separation extraction protocol
  • Collect polar and hydrophobic fractions
  • Analyze lipids and metabolites with UPLC-MS
Protocol

Biopharma Insights A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

Difficulty
intermediate
Total time
~4-6 hrs/sample
Model organism
Arabidopsis thaliana
Biosafety
BSL-1

Steps

1
Grind and disrupt tissue

Mechanically disrupt the tissue using grinding to prepare for extraction.

▶ 01:05
2
Aliquot ground tissue

Divide the disrupted tissue into aliquots for subsequent extraction steps.

▶ 02:30
3
Perform phase separation extraction

Add solvents to induce phase separation, fractionating metabolites, lipids, and proteins.

▶ 03:18
4
Aliquot polar and hydrophobic fractions

Collect and aliquote the polar and hydrophobic layers for further analysis.

▶ 04:56
5
Concentrate and analyze lipids via UPLC-MS

Concentrate the fractions and analyze the lipid content using UPLC-MS.

▶ 06:24
6
Analyze polar metabolites via UPLC-MS

Analyze the polar and semi-polar metabolite fractions using UPLC-MS.

▶ 07:48
7
Review representative chromatograms

Observe example chromatograms of lipids, metabolites, and proteins from Arabidopsis leaf extracts.

▶ 08:51
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