Improving experimental reproducibility through comprehensive research protocols

Abstract

With this “In the Limelight: Research Protocols” special issue, FEBS Open Bio aims to highlight the critical importance of publishing reproducible and detailed scientific protocols that can be broadly adopted by laboratories working in molecular and cellular life sciences. This collection includes four protocols focused on sample preparation for structural analysis of macromolecules using X‐ray crystallography, covering both in vitro and in vivo approaches. Two additional protocols demonstrate the use of cellular systems to screen the enzymatic activity of various proteins, with potential applications in high‐throughput screening. Another protocol provides a comprehensive guide for the preparation and analysis of selective autophagy flux in cultured cells using flow cytometry. Finally, the issue concludes with a protocol integrating classical behavioral tests with cognitive components to assess both physical and cognitive dimensions of frailty.

In an era of rapidly advancing technologies that are significantly impacting science, it is increasingly clear that experimentation has become more challenging. In the field of molecular and cellular life sciences, it has always been crucial to obtain trustworthy, credible, and reproducible results. However, we are now also facing the rise of fast‐track publications, which often limit researchers' ability to provide detailed explanations of their experimental procedures, data acquisition, and analysis. To address this issue, FEBS Open Bio has curated this special issue, “In the Limelight: Research Protocols,” which features a collection of detailed experimental protocols in the molecular and cellular life sciences.

First, we present the three experimental approaches for the preparation of macromolecular crystals for crystallization since resolving the atomic structures of macromolecules is of constant interest to scientists for a precise understanding of the workings of cellular processes. Claude Sauter and his team provide details on the use of a new microfluidic chip developed in their laboratory, which enables the production of crystals via the counter‐diffusion method and their direct in situ structural analysis through serial crystallography at room temperature [1].

Kaščáková et al. [2] present a protocol for the production and crystallization of protein–ligand complexes, which is essential for studying detailed protein–ligand interactions, particularly beneficial in drug discovery. In a step‐by‐step manner, they highlight both cocrystallization and ligand soaking techniques. Additionally, they introduce an alternative approach for accelerating the crystallization process through microseeding.

The third crystallography protocol switches from in vitro to in vivo crystallization: Lars Radecke and his collaborators present a detailed experimental approach to the production of crystals inside living cells [3]. The group has recently published a streamlined pipeline for producing microcrystals within insect cells, and now they provide a comprehensive guide to improve the probability of obtaining intracellular crystals from proteins recombinantly produced in insect cells [4]. They additionally provide thoughtful insight on optimizing the results.

How to produce multiprotein complexes in insect cells using a baculovirus expression system for subsequent structural analysis by crystallography is in detail provided by Bitala et al. [5]. They emphasize how selenomethionine incorporation into native proteins early in the production process is beneficial for protein analysis. They describe an effective research protocol that combines protein labeling with simultaneous production of multiproteins using coinfection of cells with monocistronic baculoviruses. Furthermore, they provide a plethora of tips and tricks on how to help the implementation of this protocol in any laboratory.

Stuparević and collaborators describe a method for implementing a tool that utilizes genetically engineered yeast cells to produce cell wall proteins capable of anchoring any enzyme of interest to investigate their unknown functions [6]. This tool transforms yeast cell walls into a living catalytic material and could serve as a high‐throughput screening platform. The system outlined in this protocol allows for precise quantification and real‐time monitoring of enzymatic activity, providing a valuable and straightforward approach for studying enzyme kinetics and protein–ligand interactions. Furthermore, the research protocol by Živković et al. [7] also focuses on enzymatic activity analysis specifically targeting the enzymes that activate substrates at the expense of ATP. To address the limitations posed by the expensive and scarce [³²P]‐PP_i, the authors developed a method that replaces it with the more accessible [³²P]‐γ‐ATP. Using aminoacyl‐tRNA synthetases as a model, the authors present an alternative that is affordable, reliable, robust, and extensively validated.

Work by Marinković et al. [8] presents the use of powerful flow cytometry to study fluorescently tagged receptors of selective autophagy. A detailed description of the preparation of cell samples for flow cytometry analysis is followed by an even more detailed postacquisition analysis. The authors emphasize that the protocol, although tested on mitophagy receptors, can be adapted to many other autophagy receptors with minor optimizations and used in laboratories with very simple flow cytometers.

Finally, Mladenović and Pracer developed a unique physical‐cognitive frailty score to measure frailty in rodents, which they bring in their research protocol [9]. Their aim is to equip researchers with improved tools for utilizing classical behavioral tests while incorporating a cognitive component to evaluate potential interventions targeting both the physical and cognitive dimensions of frailty.

I would like to express my gratitude to all the authors for their inspiring contributions to this special “In the Limelight” issue of FEBS Open Bio.

Conflict of interest

The author declares no conflict of interest.

Author contributions

IN wrote the editorial.