Jun 18, 2026

Microfractionation of a fungal culture extract

DOI
https://dx.doi.org/10.17504/protocols.io.x54v99mkmv3e/v1
  • Alice M.S. RODRIGUES1,
  • Clémence ROHÉE1,
  • Yoan FERRANDIN1,
  • Emeline HOUEL1,
  • Didier Stien1
  • 1Sorbonne Université, CNRS, UPVD, UMR8176, Laboratoire de Biodiversité et Biotechnologies Microbiennes (LBBM), Observatoire Océanologique, 66650 Banyuls-sur-Mer, France.
  • EUREMAP PROTOCOLS COMMUNITY
  • EUREMAP_SU_LBBM
Clémence ROHÉE
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DOI: https://dx.doi.org/10.17504/protocols.io.x54v99mkmv3e/v1
Protocol Citation: Alice M.S. RODRIGUES, Clémence ROHÉE, Yoan FERRANDIN, Emeline HOUEL, Didier Stien 2026. Microfractionation of a fungal culture extract. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v99mkmv3e/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 18, 2026
Last Modified: June 18, 2026
Protocol  Integer ID: 319394
Keywords: fungal culture, complex culture extract, bioactivity screening, advanced chemical annotation tool, dereplication of active microfraction, microfractionation, active microfraction, protocol for microfractionation
Funders Acknowledgements:
EUROPEAN UNION
Grant ID: INFRA-2023-DEV-01-04
Abstract
Protocol for microfractionation of a complex culture extract to facilitate bioactivity screening and dereplication of active microfractions through advanced chemical annotation tools.
Materials
HPLC- grade Solvents
DMSO (for dilution)
HPLC vials for storage
Collection tubes (minimum capacity 20 mL)
Evaporator (centrifugal or lyophilizer)
-80 °C freezer
Safety warnings
Use nitrile gloves, safety goggles and lab coat.
Before start
extraction of fungal culture completed and extract dried
Sample Preparation
Dissolve 66 mg of crude extract in 200 µL of DMSO (or any equivalent proportions to obtain a final concentration of 330 mg/mL DMSO). Inject 100 µL of this solution per fractionation cycle (equivalent to 33 mg of extract).
Chromatographic Conditions
Column maintained at 30 °C
Autosampler at 25 °C
Column: Phenomenex® Synergy™ Hydro-RP
Flow rate: 10 mL/min
UV detection at 190, 254, 280 and 310 nm or the total absorbance mode with wavelength rate from 190 to 400nm
Total fractionation time: 60 min
Fractionation:
30 tubes, 20 mL each
2 min collection per fraction
Separation gradient:
Fraction Collection and Storage
Collect 30 fractions of 20 mL each in numbered tubes.
For each fraction:
Withdraw 10% of the volume (2 mL) and transfer to an HPLC vial.
Store immediately at -80 °C
Dry the remainder of the fractions in the automatic evaporators or freezer dryer
Store the dried fractions at -80 °C
Equipments
Thermo Scientific™ Ultimate 3000 HPLC system equipped with DAD detector, column oven and fractions collector.
Automatic sample evaporators: Genevac (HT4X and EZ/2).
Freeze-dryer: Christ Beta 2-8 LO Plus