Characterisation and Analysis Services

PEF offers a number of characterisation services for analysing recombinant proteins or virus-like particles (VLPs). Services available from PEF include:

Service Brief Description
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) Separates proteins based on electrophoretic mobility. Estimate protein molecular weight and purity
Bicinchoninic acid (BCA) assay Determines total protein concentration
Western immunoblot Identifies protein using immunological specificity for amino acid sequence or epitope
Endotoxin measurement Measures endotoxin level (required for all samples destined for animal studies)
Intact protein analysis Accurately determines native protein molecular weight
Peptide mass fingerprint (PMF) Identifies purified protein or excised band from polyacrylamide gel
Analytical size exclusion chromatography (SEC-HPLC) Estimates native protein molecular weight, determine multimeric structure and degree of aggregation
Dynamic light scattering (DLS) Determines size distribution profile of proteins and protein complexes. Suitable for high throughput detection of protein aggregation, thermal stability and storage conditions
Asymmetric flow-field flow-fractionation (AF4) Accurately determines size distribution profile of VLPs and protein complexes
Transmission electron microscopy (TEM) Enables imaging and morphological characterisation of VLPs and protein complexes

For protein characterisation services, please contact us for a quote.

Training

PEF offers training for staff and students in all aspects of recombinant protein production. If you would like to enquire about training in any of the characterisation services offered by PEF, please contact us.

For an overview on the services provided by PEF, please see the PEF Production Pipeline.

Characterisation Techniques

Protein characterisation is an essential part of recombinant protein production, enabling quality control check on a purified or partially purified protein. Modern, sensitive techniques enable physical properties such as mass, size, concentration, amino acid sequence, stability and structure of a protein to be determined. Other methodologies, often protein-specific, can be used to elucidate biological functioning.

Some of the more commonly encountered characterisation techniques are described below.

Protein Quantification

The simplest method for protein quantification is measuring absorbance at 280 nm. This method does not require a dye-based kit or the preparation of a standard curve. However, the absorbance is sequence-dependent, so measurements of proteins with low tryptophan or tyrosine content may be inaccurate.

Two non-specific colorimetric assays are commonly used to obtain accurate protein concentration measurements. Both methods have different strengths and weaknesses, and the most appropriate assay will depend on the individual protein, buffer conditions and the intended application.

Bicinchoninic acid (BCA) – Proteins reduce copper ions in solution, resulting in the formation of a complex that absorbs strongly at 562 nm. Assay is linear over a broad range and is incompatible with reducing agents and chelating agents

Bradford assay – Proteins bind to Coomassie Brilliant Blue dye, resulting in a colour change in solution that can be measured at 595 nm. Assay has a relatively narrow linear range and is incompatible with detergents

Mass Spectrometry (MS)

MS is a powerful technique that can be used to determine various protein physical characteristics, including native molecular weight, sequencing information and post-translational modifications. Proteins are ionised, accelerated and separated according to their mass-to-charge ratio (m/z) under the influence of a magnetic field. As this technique is very sensitive it is possible to detect very small changes in mass e.g. a change in amino acid or chemical modification.

Analytical Size Exclusion Chromatography (SEC)

Analytical SEC can be used to elucidate protein quaternary structure. The solid phase consists of porous particles that retard the flow of smaller molecules, thereby enabling separation of proteins based on their size. The native protein molecular weight can be inferred by comparison to a reference protein, such as bovine serum albumin (BSA).

Circular dichroism (CD)

CD generates a unique spectrum based on protein secondary structure (alpha helices and beta sheets) that is used to verify correct protein folding. It is particularly useful for studying protein structure and thermodynamic stability under various environmental conditions.

Dynamic Light Scattering (DLS)

DLS is an analytical technique used to determine protein size distribution profile, and is amenable to high throughput applications. The Brownian motion of proteins in solution causes light to be scattered, with the resultant scattered intensity fluctuations dependent on particle size. Thus, average radius and the width of the distribution in terms of polydispersity can be determined. DLS can be used to quantify protein stability as a function of temperature, pH, concentration, and excipient profile.

Asymmetrical Flow Field-Flow Fractionation with Multi-angle Light Scattering (AF4-MALS)

The AF4-MALS technique offers high-resolution biophysical characterisation of large protein complexes and virus like-particles (VLPs). Complexes are separated based on their size, followed by MALS detection of each component’s dynamic properties. AF4-MALS is commonly applied to detect batch-to-batch variation, degradation or aggregation during VLP formulation and storage.

Transmission electron microscopy (TEM)

TEM is used for morphological characterisation of protein complexes or VLPs. A beam of electrons is transmitted through a fixed protein sample, enabling large-scale protein structures to be imaged. This technique is often used in conjunction with AF4-MALS for VLP characterisation.

Related Articles on Protein Characterisation

Wu Z,  et al. Structural Characterization of a Recombinant Fusion Protein by Instrumental Analysis and Molecular Modeling. PLoS ONE. 8(3), e57642 (2013) Link

Jachimska B, et al. Characterization of Globular Protein Solutions by Dynamic Light Scattering, Electrophoretic Mobility, and Viscosity Measurements. Langmuir. 24(13), 6866-6872 (2008) Link

Wen J, et al. Size-Exclusion Chromatography with On-Line Light-Scattering, Absorbance, and Refractive Index Detectors for Studying Proteins and Their Interactions. Analytical Biochemistry. 240(2), 155-166 (1996) Link