Chapter 1
Assessing and Improving Protein Sample Quality
Bertrand Raynal, Se ´ bastien Bru ˆ le ´ , Stephan Uebel, and Stefan H. Knauer
Abstract
One essential prerequisite of any experiment involving a purified protein, such as interaction studies or
structural and biophysical characterization, is to work with a “good-quality” sample in order to ensure
reproducibility and reliability of the data. Here, we define a “good-quality” sample as a protein preparation
that fulfills three criteria: (1) the preparation contains a protein that is pure and soluble and exhibits
structural and functional integrity, (2) the protein must be structurally homogeneous, and (3) the preparation must be reproducible. To ensure effective quality control (QC) of all these parameters, we suggest
to follow a simple workflow involving the use of gel electrophoresis, light scattering, and spectroscopic
experiments. We describe the techniques used in every step of this workflow and provide easy-to-use
standard protocols for each step.
Key words Purity, Homogeneity, Identity, Oligomeric state, Structural integrity, Protein stability,
Optimization of storage conditions, Batch-to-batch consistency
1 Introduction
The overall quality control (QC) workflow consists of a series of
experiments that are intended to assess the purity, structural homogeneity, and stability of a protein sample (Fig. 1). The data obtained
can demonstrate, inasmuch as is reasonably practicable, that the
sample is of good quality, can be used to verify batch-to-batch
consistency of protein preparations, or, where relevant criteria are
not met, can be used to guide iterative improvement of the quality
of protein samples.
Prior to any experimental work, it is essential to gather all
information about the sample that is necessary for the setup of
experiments and data analysis. Researchers should know the full
amino acid sequence of their target protein (including tags, additional residues from cleavage sites, etc.) and the identity of all other
known chemical components in the sample (i.e., those derived from
its preparative process). Full information on the safety of the protein and other components of the sample should be obtained as this
can have an impact on the practical performance of experiments.
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
3
Assessing and Improving Protein Sample Quality
Bertrand Raynal, Se ´ bastien Bru ˆ le ´ , Stephan Uebel, and Stefan H. Knauer
Abstract
One essential prerequisite of any experiment involving a purified protein, such as interaction studies or
structural and biophysical characterization, is to work with a “good-quality” sample in order to ensure
reproducibility and reliability of the data. Here, we define a “good-quality” sample as a protein preparation
that fulfills three criteria: (1) the preparation contains a protein that is pure and soluble and exhibits
structural and functional integrity, (2) the protein must be structurally homogeneous, and (3) the preparation must be reproducible. To ensure effective quality control (QC) of all these parameters, we suggest
to follow a simple workflow involving the use of gel electrophoresis, light scattering, and spectroscopic
experiments. We describe the techniques used in every step of this workflow and provide easy-to-use
standard protocols for each step.
Key words Purity, Homogeneity, Identity, Oligomeric state, Structural integrity, Protein stability,
Optimization of storage conditions, Batch-to-batch consistency
1 Introduction
The overall quality control (QC) workflow consists of a series of
experiments that are intended to assess the purity, structural homogeneity, and stability of a protein sample (Fig. 1). The data obtained
can demonstrate, inasmuch as is reasonably practicable, that the
sample is of good quality, can be used to verify batch-to-batch
consistency of protein preparations, or, where relevant criteria are
not met, can be used to guide iterative improvement of the quality
of protein samples.
Prior to any experimental work, it is essential to gather all
information about the sample that is necessary for the setup of
experiments and data analysis. Researchers should know the full
amino acid sequence of their target protein (including tags, additional residues from cleavage sites, etc.) and the identity of all other
known chemical components in the sample (i.e., those derived from
its preparative process). Full information on the safety of the protein and other components of the sample should be obtained as this
can have an impact on the practical performance of experiments.
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
3