Experiment: Protein Purification and Assays
Careful preparation ensures experimental success. Whether you are performing time-course experiments, analyzing protein expression, or performing assays for drug development, all of your tools must perform optimally to generate trustworthy data.
To help you move from the hood to the bench, we offer an array of technologies for purifying proteins and looking at cellular activity.
If the purpose of your cell culture is protein purification, a large part of your day is likely spent filtering, purifying, and concentrating your protein. These steps are challenging because the protein can be degraded, precipitated, or otherwise lost at any step during a multi-day purification protocol. Selecting the right tools can improve your efficiency and decrease the chance of protein loss.
Every step of your protocol can chip away at your protein yield:
Protein degradation during cell lysis. It can take hours to lyse your cells and clarify the supernatant, so your protein is exposed to proteases that can truncate or completely degrade it.
Drying out during concentration. After a successful purification step, concentration may seem trivial. But in the absence of built-in precautions, you may lose your protein in the protein concentrator by spinning your sample too fast or for too long.
Leakage during dialysis. During the long, multi-step dialysis, your protein could leak from the dialysis bag or cassette. Even if the cassette is fully intact, your protein can unfold, refold, aggregate, or undergo proteolysis, especially in a multi-day protocol. It is especially frustrating to lose protein at this final stage.
Solutions to keep your protein intact and your purification fast:
Application Note
Sartorius Ultrafiltration Products in the Preparation of Biological and Medical Nanocarriers – a Short Review
Working with secreted proteins allows you to avoid the messy lysis process. In addition, your protein never encounters intracellular proteases during purification. Regardless of whether your protein is naturally secreted or you have optimized your intracellular protein to allow purification in HEK Freestyle or another optimized cell line, with a secreted protein, you encounter a different set of challenges. For instance, intact cells must be separated from a relatively large volume of supernatant and the protein sample must be concentrated.
Purifying secreted proteins
If you culture cells for production of secreted proteins, then a large part of your process is purifying and concentrating your protein. To shorten your long days of protein purification, we offer a kit that allows you to clarify cell culture supernatant in a single step. This innovative kit simplifies your protein purification process by fully eliminating centrifugation steps and decreasing your time to clarified supernatant from hours to minutes.
Tools to rapidly isolate supernatant with your secreted protein:
Sartoclear Dynamics Lab Cell Culture Clarification System
Trust in Your Equipment When Harvesting Cell Culture Supernatant
Find out why IBA Scientist, Dennis Karthaus in Protein Products & Assay Department trusts Sartoclear Dynamics Lab to save time and money when harvesting cell culture supernatants.
Application Note
From Mammalian Cell Cultures to Pure Proteins: Sartoclear Dynamics® Lab Significantly Reduces Cell Harvest Time
Application Note
Lab-Scale Clarification of Mammalian Suspension Cultures Using Sartoclear Dynamics® Lab V Kits
Gain cellular insights precisely and quickly. If your goal is to provide physiologically relevant information about your cells with reduced variability, you need the right imaging and analysis tools. The best tools provide reproducible and information-rich analyses to help you quickly gain insights and cost-effectively publish your results or move forward with additional assays.
Technology for live-cell analysis
Cellular Proliferation
Proliferation is an essential mechanism for normal tissue development, regeneration, and renewal. The three main types of biochemical cell proliferation assays are based on DNA synthesis, metabolic activity, and ATP concentration, and are typically sequential, single-endpoint assays coupled together to generate time course data. These endpoint measurements are indirect and subject to artifacts that cannot be readily verified by morphological changes.
Live-cell imaging allows the continuous measurement of cell proliferation assays right inside your incubator, such as label-free confluence measurements and direct cell counting using fluorescent labeling.
Apoptosis
Apoptosis is an essential process for normal tissue development and homeostasis, in which cells undergo timely programmed cell death. Common assays for apoptosis include enzymatic assays to measure caspase-3/7 activation or PS externalization using plate readers or flow cytometers. These common assays yield a single, user-defined end-point measurement, require multiple wash steps or cell lifting that may result in artifacts, and are not amenable to long-term measurements due to increasing background signal over time.
Live-cell analysis uses specialized reagents to measure multiple apoptotic pathways both simultaneously and in real time. Apoptotic signals can then be correlated with phase contrast images to provide additional biological insight and morphological validation of apoptotic cell death.
Cytotoxicity
Cytotoxicity is a general term that describes the detrimental effects of substances or environmental changes on cell health that may compromise metabolic activity, inhibit cell growth or division, or ultimately cause cell death.
A number of cytotoxicity assays involve the measurement of cell membrane integrity, either with dyes (to which healthy cells are impervious) or via the release of markers from dying cells. Metabolic activity measurements are also used to measure cell cytotoxicity. However, few of these assays involve direct counting of dying cells over time.
Live-cell analysis can elucidate cytotoxic pathways based on cell membrane integrity in real time. The nuclei of dying cells take up an inert dye excluded from live cells, and dying cells are identified over time by an increase in fluorescent signal. Cytotoxic signals can then be correlated with phase contrast images to provide additional morphological validation of cell death.
High-throughput live-cell imaging and analysis. Measurements of cell health, such as proliferation, apoptosis, and cytotoxity, are essential when studying the effects of candidate compounds on cell growth and viability, allowing you to rank candidate compounds, identify off-target compounds, and help determine mechanism of action. Real-time, live-cell analysis has considerable advantages over end-point assays when evaluating drug candidates. For instance, using real-time, live-cell analysis, it is possible to determine the effective concentration of a candidate compound and the time needed to perturb the target in a single assay, allowing you to accelerate your candidate screening pipeline.
With real-time, live-cell analysis, images of cell cultures in 96- or 384-well plates can be automatically acquired and analyzed to generate time-courses and see concentration-dependent responses for calculating EC50s or IC50s.
Cell Culture Quality Control
Variability in your cell culture workflows can adversely affect the downstream consistency and reproducibility of your drug development program. Real-time, live-cell analysis allows you to monitor your cells without the need to remove them from the incubator, thus automating data capture and cell assessment. Cells are monitored in precise intervals, and the objective, image-based data may be archived for comparison with current cell growth months or years later.
