Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Representation and validation of the AF4‐MALS‐FLD method . (A) Overview of the workflow used for identification of EV surface proteins. PE‐conjugated antibodies were incubated with the sample (e.g. pre‐purified EVs, cell culture supernatant, urine, or plasma) and loaded into the AF4 channel. (B) The light scatter elution profile (in relative scale) (black, full line), UV elution profile (black, dotted line) and the size determination ( R rms in nm) (red) obtained by the multi‐angle light scattering (MALS) detector is plotted against time for labelling of SK‐BR‐3‐derived EVs with PE‐conjugated anti‐CD81 antibody. (C) The fluorescent light detector (FLD) signal (in relative scale) for SK‐BR‐3‐derived EVs labelled with PE‐conjugated anti‐CD9, anti‐CD63 and anti‐CD81 is plotted against time. (D) Transmission electron microscopy (TEM) images of different fractions of the AF4‐MALS‐FLD elution profile are shown (scale bar = 200 nm).

Article Snippet: Detection was performed by an ultraviolet (UV) detector at 280 nm (Shimadzu), a multi‐angle light scattering (MALS) detector DAWN HELEOS‐II using a laser at 658 nm (Wyatt Technology) and a fluorescence detector (FLD) with an excitation at 488 nm, emission at 578 nm and a gain of 16 (Agilent Technologies) (Geeurickx et al. ).

Techniques: Biomarker Discovery, Incubation, Purification, Cell Culture, Clinical Proteomics, Multi-Angle Light Scattering, Derivative Assay, Transmission Assay, Electron Microscopy

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: AF4‐MALS‐FLD analysis of EV surface proteins with biomarker potential in prostate and breast cancer . MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. (A) The elution profile (in relative scale) of the multi‐angle light scatter (MALS) detector and the size ( R rms in nm) were plotted against time. The fluorescent light detector (FLD) signal for MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs labelled with (B) PE‐conjugated anti‐EpCAM and (C) PE‐conjugated anti‐HER2 antibodies were plotted. (D) From FLD elution profiles, the area under the curve for the EV peak (24–80 min) was determined. Unstained EV samples were used as a negative control. (E) Different concentrations (6 × 10 9 , 8 × 10 9 , 1 × 10 10 and 2 × 10 10 particles as measured by NTA) including a negative control of LNCaP‐derived EVs (high PSMA expression) were labelled with anti‐PSMA antibodies and analysed by the AF4‐MALS‐FLD protocol. (F) The area under the curve for the EV peak was determined for LNCaP‐derived EVs. Different concentrations (2 × 10 10 , 4 × 10 10 and 6 × 10 10 particles as measured by NTA) including a negative control of (G) MCF‐7‐derived EVs (high EpCAM expression) or (I) SK‐BR‐3‐derived EVs (high HER2 expression) were labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively and analysed by the AF4‐MALS‐FLD protocol. The area under the curve for the EV peak (24–80 min) was determined for (H) MCF‐7‐ and (J) SK‐BR‐3‐derived EVs.

Article Snippet: Detection was performed by an ultraviolet (UV) detector at 280 nm (Shimadzu), a multi‐angle light scattering (MALS) detector DAWN HELEOS‐II using a laser at 658 nm (Wyatt Technology) and a fluorescence detector (FLD) with an excitation at 488 nm, emission at 578 nm and a gain of 16 (Agilent Technologies) (Geeurickx et al. ).

Techniques: Biomarker Discovery, Derivative Assay, Multi-Angle Light Scattering, Negative Control, Expressing

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Detection of EVs in complex matrices . (A) Different volumes of cell culture supernatant (0, 20, 40 and 60 µL) collected from the MCF‐7 cells were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak in complex matrices (40–80 min) was determined. (B) Different amounts of LNCaP‐derived EVs were spiked in 100 µL of concentrated urine, diluted 1:1 in PBS to reduce viscosity, labelled with PE‐conjugated anti‐PSMA antibodies, and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak was determined. Different amounts of (C) MCF‐7‐ or (D) SK‐BR‐3‐derived EVs were spiked in 100 µL of blood plasma, diluted 1:1 in PBS to reduce viscosity, and labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies, respectively. Labelled EVs were analysed by AF4‐MALS‐FLD and the area under the curve for the EV peak was determined. Different amounts of SK‐BR‐3 EVs were also spiked in blood plasma and labelled with isotype control antibodies. (E) Different concentrations of soluble EpCAM (1, 5 and 10 ng/mL) and soluble HER2 (50, 100 and 150 ng/mL) were spiked in blood plasma, labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively, and analysed by AF4‐MALS‐FLD.

Article Snippet: Detection was performed by an ultraviolet (UV) detector at 280 nm (Shimadzu), a multi‐angle light scattering (MALS) detector DAWN HELEOS‐II using a laser at 658 nm (Wyatt Technology) and a fluorescence detector (FLD) with an excitation at 488 nm, emission at 578 nm and a gain of 16 (Agilent Technologies) (Geeurickx et al. ).

Techniques: Cell Culture, Derivative Assay, Viscosity, Clinical Proteomics, Control

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Validation of the AF4‐MALS‐FLD workflow on patient samples . Urine samples of five prostate cancer patients were labelled for PSMA and analysed by the AF4‐MALS‐FLD workflow. Fractions 40–80 min were collected, concentrated and processed for mass spectrometry‐based proteomic analysis. (A) EV markers Syntenin‐1, Flotillin‐1, CD63, CD9, CD81, Flotillin‐2, Alix and TSG101 were analysed (missing sample indicated in grey). Z ‐score transformation of intensities were plotted. (B) Targeted mass spectrometry analysed the presence of PSMA (FOLH1) in patient samples. The z ‐score transformation of intensities was plotted with the AF4‐MALS‐FLD peak area. (C) Blood plasma samples of healthy controls ( n = 7) and HER2 amplified breast cancer patients ( n = 10) were labelled with PE‐conjugated anti‐HER2 antibodies. (D) Blood plasma samples of healthy controls ( n = 6) and breast cancer patients ( n = 8) were labelled with PE‐conjugated anti‐EpCAM antibodies. The area under the curve values were normalised for the mean value in the healthy control group.

Article Snippet: Detection was performed by an ultraviolet (UV) detector at 280 nm (Shimadzu), a multi‐angle light scattering (MALS) detector DAWN HELEOS‐II using a laser at 658 nm (Wyatt Technology) and a fluorescence detector (FLD) with an excitation at 488 nm, emission at 578 nm and a gain of 16 (Agilent Technologies) (Geeurickx et al. ).

Techniques: Biomarker Discovery, Mass Spectrometry, Transformation Assay, Clinical Proteomics, Amplification, Control

Journal: bioRxiv

Article Title: The HMD domain of the PAF complex primes Rad6-Bre1 E3 ligase complexes for H2B ubiquitination

doi: 10.64898/2026.03.01.708808

Figure Lengend Snippet: (A) Domain structure of S. pombe HULC subunits. CC: coiled coil; RING: really interesting new gene domain. RBD: Rad6-binding domain, IDR: intrinsically disordered region, UBC: Ubiquitin C E2 ligase domain (B) SDS PAGE gel of size exclusion peak fraction of HULC and western blot against T7-tagged Brl1, Shf1 and Rhp6 after StrepII-tag pull-down of HULC. (C) Mass photometry size distribution of affinity-purified HULC. (D) Size exclusion coupled with multi-angle light scattering (MALS) showing UV chromatogram overlaid with molecular mass determination for HULC peak. (E) Sedimentation velocity c(S) distribution of HULC after size exclusion chromatography (see fit in Fig. S2). (F) Spot assay of indicated S. pombe strains on yeast extract agar (YE). (G) Quantification of red colony frequencies for the mutant strains used in (F). All solution experiments were in 20 mM HEPES pH 7.5, 150 mM KCL, 2 mM MgCl 2 .

Article Snippet: Multi-angle light scattering was determined by in-line measurement of the Superose 6 column elution using an Optilab T-rEX differential Refractive index detector connected to a DAWN HELEOS MALS detector (Wyatt Technology).

Techniques: Binding Assay, Ubiquitin Proteomics, SDS Page, Western Blot, Affinity Purification, Multi-Angle Light Scattering, Sedimentation, Size-exclusion Chromatography, Spot Test, Mutagenesis