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Centrifugal devices are used in a wide variety of industries for protein preparation, including for protein concentration, buffer exchange, and fractionation of proteins and nucleic acids. A concentration gradient can often occur during centrifugation due to the sample reservoir design, which leads to excessively high sample concentrations at the bottom of the reservoir. This affects the biological activity of a protein, preventing its use for the intended analytical purpose. Innovative spin device designs have been developed to minimize or potentially eliminate the incidence of aggregation, and are critical to the ongoing research of many laboratories.
Protein aggregation can occur when a protein denatures and unfolds, exposing previously concealed hydrophobic sites that bind to hydrophobic regions on other molecules, or when native proteins form aggregates via hydrophobic regions on their outer surface. The formation of aggregates depends on the solution conditions, temperature, and protein concentration. Avoiding these conditions often demands extra protein purification steps, driving up costs.
Downstream processing for the recovery and purification of proteins is achieved by exploiting the physiochemical properties of molecules and aggregates, such as size, charge, and shape. There are various methods used to do this, such as chromatography, centrifugation, filtration, and precipitation. Many laboratories use centrifugal devices to support their work, particularly in cancer, biochemical, and pharmacology research. In pharmacology applications, scientists aim to improve the efficacy of drugs and determine how to deliver them to the target site without causing systemic side effects.
The first step after protein extraction is protein purification. Conventional methods require centrifugation to pellet the sample, followed by removal of the buffer and recovery of beads. Here, it is important to minimize sample loss. Before downstream analysis, many proteins must be concentrated for high-quality data output and reproducible protein assay results. The protein must be in its native, soluble form, dissolved in the buffer at an appropriate concentration, without compromising the yield, even if the protein is low in abundance.
Studying the proteome
Western blotting is a central tool for large-scale proteomics studies to detect the presence of proteins in unknown or complex sample mixtures and determine the relative abundance of certain proteins. Once proteins have been separated from a sample via gel electrophoresis, they are transferred to a specific membrane, which immobilizes them for downstream analysis. After incubation with labeled antibodies to identify the protein of interest, the membrane is washed to remove any unbound antibodies and is further analyzed using immunodetection methods.
The need to analyze specific proteins from increasingly small samples has driven the development of novel centrifugal devices. Many academic laboratories perform primary Western blots multiple times per week in order to visualize the protein on a membrane and to find out how far the proteins express. Concentrating proteins with a higher input can be challenging. Centrifugal devices for difficult-to-concentrate proteins, such as membrane proteins, can be used to quantify and compare expression among experimental groups using a small sample size.
Purifying small sample sizes
All clinical and proteomics research laboratories aim to reduce the number of protein purification steps. Sample preparation is vital in proteomics research, and ineffective preparation can lead to loss of valuable samples and time, while effective steps can facilitate the discovery of new therapeutics. As samples get smaller and more numerous, the need for novel methods to purify proteins and improve assays intensifies, leading to the production and development of innovative centrifugal devices.
Patient samples frequently lack sufficient protein for Western blot analysis in pharmacological research, which can be the greatest limitation of this technique: it can be difficult to visualize rare expressing proteins. To avoid the complications of a small sample volume, experiments can begin with a higher amount of protein. In real-life applications, however, researchers can rarely request a larger patient sample once it has been obtained, because they often are taken during surgery or clinical visits. For example, tumor samples are usually between 2 and 3 mg, and from these the protein must be extracted, but there is not enough expression of the protein of interest. A larger tissue sample cannot be obtained once the procedure is complete, but the sample preparation step can be adapted so that it is compatible with the experimental design. For example, Western blot is not sensitive enough to detect 1 μg for every 25 μg of total protein, but concentrated protein can be used with the correct molecular-weight cut-off (MWCO) and eliminate all the uninterested proteins and salts to counter this. Smaller centrifugal devices are therefore useful.
Ultrafiltration is an alternative to Western blot. As a membrane separation technique that uses molecule size as the primary basis for separation, it separates very small particles and dissolved molecules in fluid. Molecules larger than the membrane pores are retained by the membrane surface and are concentrated. Molecule shape and charge can also play a role in membrane retention. For example, linear molecules such as DNA will be able to pass more easily though pores that are designed to retain globular-shaped proteins of the same molecular weight. Ultrafiltration allows the removal or exchange of salts and the removal of detergents (diafiltration) and is popular because it is usually relatively rapid and does not adversely affect the protein sample.
A time-saving workflow
Protein purification is a complex process that involves a number of intricate sequential steps. The separation step usually creates the need for the sample to be desalted or concentrated to prepare the biomolecule sample for the next step of purification. Using ultrafiltration, molecules are separated based on molecular size, so laboratories can choose the appropriate device based on the MWCO. Most centrifugal devices are suitable for 4–15 mL samples, but many laboratories are realizing the benefits of smaller (1.5–2 mL) devices, as this is more practical in the basic science laboratory, especially for Western blot applications, where concentrated protein is not always available.
By using concentrated proteins at smaller sample sizes, the experiment does not need to be repeated as it would with nonconcentrated proteins. This saves the laboratory valuable time and allows results to be visualized more quickly.
The majority of spin device designs excessively concentrate the protein solution where the filters are located due to the “V” shape of the funnel insert. Excessive concentration creates a protein gradient that is very dense at the bottom of the filter, thereby drastically increasing the risk of aggregation and precipitation, and ultimately affecting the biological activity of a protein. Some devices have a circular (“U”-shaped) sample reservoir and include the filter unit. This provides a wider area to disperse the concentrated protein in a more efficient manner. The traditional conical shape of the device (Figure 1, right) can make it challenging to resuspend samples. This is made easier by the flat shape of smaller devices (left), which also reduce the chance of damaging the membrane with the pipette tips.
Figure 1 – Left: 1-kDa MWCO concentrators with “U”-shaped sample reservoir. Right: Industry-standard peptide concentrators with “V”-shaped reservoir.Two case studies illustrate these devices in use. One describes the devices used by Harshul Pandit, graduate Ph.D. student from the University of Louisville, to analyze specific proteins from increasingly small samples.1
The second case study highlights the work of a laboratory that uses nuclear magnetic resonance (NMR) spectroscopy to investigate macromolecular targets and obtain an atomic-level understanding of biological molecules and their interactions.2 To overcome the challenges to NMR of using large molecules, the group uses specialized centrifugal devices to obtain the individual peptides for protein interaction studies, to achieve the exact length of peptide required for optimized NMR analysis.
References
- http://go.pall.com/centrifugation-pharmacology-research.html?
- http://go.pall.com/centrifugal-devices-cancer-research.html?utm-source
Tom Valorose is global product manager—Analytical, Pall Laboratory, 20 Walkup Dr., Westborough, MA01581, U.S.A.; tel.: 508-871-5481; e-mail: [email protected]; www.pall.com