A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies

Štrancar, Urša; D’Ercole, Claudia; Cikatricisová, Lucia; Nakić, Mirna; De March, Matteo; de Marco, Ario

doi:10.3390/biom14050587

Open AccessArticle

A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies

by

Urša Štrancar

^†

,

Claudia D’Ercole

^†

,

Lucia Cikatricisová

,

Mirna Nakić

,

Matteo De March

and

Ario de Marco

^*

Laboratory of Environmental and Life Sciences, University of Nova Gorica, Vipavska cesta 13, Rožna Dolina, 5000 Nova Gorica, Slovenia

^*

Author to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Biomolecules 2024, 14(5), 587; https://doi.org/10.3390/biom14050587

Submission received: 9 April 2024 / Revised: 7 May 2024 / Accepted: 14 May 2024 / Published: 15 May 2024

(This article belongs to the Special Issue Conventional and In Silico-Based Approaches for the Optimization of Nanobody Biophysical Characteristics)

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Abstract

Background: Fluorescent proteins (FPs) are pivotal reagents for flow cytometry analysis or fluorescent microscopy. A new generation of immunoreagents (fluobodies/chromobodies) has been developed by fusing recombinant nanobodies to FPs. Methods: We analyzed the quality of such biomolecules by a combination of gel filtration and SDS-PAGE to identify artefacts due to aggregation or material degradation. Results: In the SDS-PAGE run, unexpected bands corresponding to separate fluobodies were evidenced and characterized as either degradation products or artefacts that systematically resulted in the presence of specific FPs and some experimental conditions. The elimination of N-terminal methionine from FPs did not impair the appearance of FP fragments, whereas the stability and migration characteristics of some FP constructs were strongly affected by heating in loading buffer, which is a step samples undergo before electrophoretic separation. Conclusions: In this work, we provide explanations for some odd results observed during the quality control of fluobodies and summarize practical suggestions for the choice of the most convenient FPs to fuse to antibody fragments.

Keywords: fluorescent proteins; fluobodies; protein degradation; alternative start codons

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MDPI and ACS Style

Štrancar, U.; D’Ercole, C.; Cikatricisová, L.; Nakić, M.; De March, M.; de Marco, A. A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies. Biomolecules 2024, 14, 587. https://doi.org/10.3390/biom14050587

AMA Style

Štrancar U, D’Ercole C, Cikatricisová L, Nakić M, De March M, de Marco A. A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies. Biomolecules. 2024; 14(5):587. https://doi.org/10.3390/biom14050587

Chicago/Turabian Style

Štrancar, Urša, Claudia D’Ercole, Lucia Cikatricisová, Mirna Nakić, Matteo De March, and Ario de Marco. 2024. "A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies" Biomolecules 14, no. 5: 587. https://doi.org/10.3390/biom14050587

APA Style

Štrancar, U., D’Ercole, C., Cikatricisová, L., Nakić, M., De March, M., & de Marco, A. (2024). A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies. Biomolecules, 14(5), 587. https://doi.org/10.3390/biom14050587

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

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A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies

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