Ensuring the production or extraction of high-quality protein molecules requires multiple quality control (QC) steps to confirm the identity of the final product and detect any impurities. Historically, such QC has been accomplished using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the time-consuming and error prone nature of SDS-PAGE has led to the development of automated systems for analyzing protein samples. Automated platforms offer a wealth of advantages, including shorter analysis times and higher sensitivity than traditional methods, and have evolved to handle challenging sample types such as crude lysates and high-salt purification fractions.
Purified proteins are essential for a broad range of applications. They are used as controls in immunoassays, such as western blot and ELISA, and can serve as capture molecules in antibody tests. Depending on the intended application, the protein of interest may be extracted directly from a cell/tissue lysate or be recombinantly expressed. In either scenario, some form of purification is generally required prior to use.
For low resolution purification, methods such as ammonium sulfate precipitation (an approach based on protein solubility in salt solutions of varying saturation) may suffice. However, it is more common for proteins to be purified according to properties such as size, charge, or molecular recognition using various chromatography methods (e.g., size exclusion, ion exchange, and affinity chromatography, respectively). To determine the identity and purity of proteins that have been isolated in this way, it is necessary to analyze the cell expression lysate, binding flow through, washes, and elution fractions.
One of the most widely used methods for monitoring protein purification is SDS-PAGE, which separates proteins according to their molecular weight. A typical protocol involves preparing samples in a buffer containing SDS, which binds proteins in proportion to their relative molecular mass and imparts a negative charge. The samples are then loaded onto a polyacrylamide gel alongside a protein ladder. Upon applying a charge to the gel, the proteins migrate toward the positive electrode, with smaller proteins migrating fastest. Lastly, the gel is stained with Coomassie blue, followed by incubation in a destain solution, and imaged as required. A major disadvantage of SDS-PAGE is the length of time to results, which can be up to a day. Additionally, SDS-PAGE offers only limited resolution and is error prone due to its manual workflow, which includes the need to annotate data by hand.
Automated systems for analyzing protein samples are generally based on sodium dodecyl sulfate capillary electrophoresis (CE-SDS)—a method analogous to SDS-PAGE, but which involves running samples through a gel-filled capillary for analysis via UV or fluorescent detection. Importantly, automated CE-SDS offers many advantages over traditional methods. First, the analysis time is much shorter. Platforms such as the ProteoAnalyzer System* from Agilent Technologies can simultaneously analyze 12 protein samples in as little as 30 minutes. Automated CE-SDS also provides higher separation resolution, making it possible to resolve glycosylated and non-glycosylated protein isoforms. Other advantages of CE-SDS include its superior sensitivity and quantitative linear dynamic range, which allows for detecting low level impurities, and its capacity to generate quantitative digital data. Additionally, sample requirements are reduced to as little as 1 µL, while experimental reproducibility is enhanced by eliminating the need for laborious manual processing.
Until relatively recently, automated CE-SDS platforms struggled to accurately analyze certain sample types, most notably crude lysates and high-salt purification fractions. Yet these challenges have been overcome with key technological advances.
Figure 1 shows sizing and expression data verifying synthesis of the calmodulin-like 3 (CALML3) protein in a cell-free production system. Panel A, generated using the Agilent ProteoAnalyzer, highlights the ability of the automated system to quickly overlay the CALML3 expression sample and its negative control, allowing for % expression to be determined. Panel B shows SDS-PAGE data confirming protein expression.
Figure 1. Analysis of crude protein lysate from a cell-free expression system for CALML3. (A) Electropherogram overlay and digital gel image of CALML3 protein expression assessed by the Agilent ProteoAnalyzer system. (B) SDS-PAGE results. (C) Comparison of size and percent expression.
Compared to traditional methods for monitoring protein purification, such as SDS-PAGE, automated systems offer many advantages. These include shorter analysis times and higher separation resolution, even for challenging sample types.
The Agilent ProteoAnalyzer system is an automated capillary electrophoresis instrument designed for the assessment of protein samples including monoclonal antibodies, biosimilars, crude lysates, and protein purification fractions. To learn more, visit agilent.com/proteoanalyzer-systems
*For Research Use Only. Not for use in diagnostic procedures. PR7001-4069