Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Modern biology would be a lot more difficult without ways of separating the different compartments and components that make up the cell. From cell signaling to metabolism, it’s important to know where things are, where they’ve been and where they’re going.
Researchers fractionate cells for many reasons, says Monica O’Hara Noonan, market segment manager for Thermo Fisher Scientific. They may want to track a protein of interest as it translocates under certain conditions or following certain treatments. Fractionating the cell can help enhance detectability of low-abundance proteins, she says, or reduce the complexity for downstream analyses or functional assays.
If you find yourself pursuing such research directions, read on. We’ll help you separate the wheat from the chaff, cellularly speaking.
Fractions of a cell
How to fractionate a cell depends on many factors, not the least of which are available equipment, expertise, what you’re trying to isolate and the downstream use to which the fractions will be put.
Centrifugation of one type or another is the most common technique for cell fractionation, either alone or in combination with other methods [1]. For organelles such as the Golgi apparatus or peroxisomes, for example, most protocols call for density-gradient centrifugation at high speeds. Yet ultracentrifugation has a few drawbacks, points out O’Hara Noonan, including that ultracentrifuges are big-ticket items to which some labs lack access. Another difficulty: Density gradients are notoriously difficult to pour and run, with the results very technique-dependent.
Many other fractions, though, can be enriched using only reagents and benchtop equipment. Vendors have developed kits that can quickly and easily deliver products highly enriched for a particular compartment of a cell. Thermo Fisher’s newly revamped Mem-PER Plus kit, for example, allows integral and membrane-associated proteins to be enriched in about an hour.
The principle behind such kits is for the most part comparable across vendors, says Michael Moehlenbrock, product specialist at Sigma-Aldrich. They tend to use a hypotonic buffer to initially stress the cell membrane, and isotonic extraction buffer—with or without reducing agents and protease inhibitors—to maintain the organelles of interest. Mechanical lysis (using a Dounce homogenizer) or detergent lysis sometimes is called for, as well. “There is consistency in the design of cell-fractionation kits across commercial vendors, and differentiation between kits for different cellular fractions is based on buffer composition, lysis reagent and centrifugation speeds,” Moehlenbrock says.
The value of such kits is not in some black-box reagent, but the consistency and reproducibility of QC’d systems. "Cell-fractionation methods have been well characterized and protocols are well known for most applications,” Moehlenbrock says. “Commercial kits allow for simplicity and efficiency for the end user while providing them the security of quality assurance."
By varying the detergent compositions in the buffers, interspersed with centrifugation steps, researchers can isolate more than one distinct fraction. Many kits on the market collect, for example, both nuclear and cytosolic-protein fractions. Others deliver three or four different fractions—Cell Signaling Technology’s Cell Fractionation Kit results in cytosolic, membrane/organelle and nuclear/cytoskeletal compartments, for example, and Qiagen’s Qproteome Cell Compartment Kit separates the cytosolic, membrane, nuclear and cytoskeletal compartments.
Miltenyi Biotec offers a detergent-free kit that uses anti-TOM22-labeled magnetic microbeads to specifically isolate mitochondria. The company does not offer products for other subcellular fractions, according to a technical support representative, but customers have used the company’s products to indirectly isolate other fractions using their own antibodies.
Similarly, Thermo Fisher Scientific’s Cell Surface Protein Isolation Kit labels the external lysines of cell-surface proteins with a cleavable biotin derivative, allowing this protein fraction to be captured after the cells are lysed.
What’s in there?
After the fractions are isolated, it’s important to validate your experiment and verify that the fractions actually contain what you set out to isolate. Although the kits generally don’t contain reagents that will differentiate one fraction from another, many will point out “a number of different housekeeping genes that you could use to ensure quality,” says Moehlenbrock. “And in some cases, we provide suggestions on assays.” For example, the technical bulletin for Sigma-Aldrich’s Mitochondria Isolation Kit suggests looking at cytochrome c oxidase activity to check outer-membrane integrity and citrate synthase activity for inner-membrane integrity.
Cell Signaling Technology offers its Cell Fractionation Antibody Sampler Kit as a companion to its fractionation kit, “so that when you run a Western, you can get a quantifiable number for which fraction your protein is in,” says production scientist Jim Cormier. The Sampler Kit contains rabbit monoclonal antibodies against cytoplasmic MEK1/2, mitochondrial apoptosis-inducing factor (AIF), nuclear histone H3 and cytoskeletal vimentin, along with a labeled goat anti-rabbit IgG. “You’re validating against a standard, so that if you do the experiment again next week, you can be sure the results are again validated against the same standard.”
Researchers also can use the recommendations found in the kits or do a literature search and assemble reagents of their own for Western blots or ELISA kits. But be advised that that “not every antibody will have the kind of specificity it ought to have,” Cormier cautions.
Upstream prep for downstream
Reagent-based methods for fractionating cells are fast and convenient, especially when compared with ultracentrifugation, and can feed a host of downstream applications, such as electrophoretic mobility shift (“gel shift”) assays, reporter assays, enzyme activity assays and Western blotting, for example.
Yet the detergents and salts used in the preparations can be anathema to proteomic applications like mass spectrometry, and “sometimes it depends on the amount of the detergent, and how easy it is to remove, whether the prep can be used for [a] functional assay,” says O’Hara Noonan. “In some cases, you may have to do dialyses or desalting.”
Similarly, extra steps may be necessary to increase a prep’s purity. As a graduate student, Moehlenbrock routinely prepared mitochondria using a tabletop centrifuge. But “there were times when I wanted to get out additional cell debris that would come along with the 11,000 x g spin down. Then I would take my mitochondrial isolate and further clean it up using a sucrose gradient and ultracentrifuge,” he recalled. “It just depends on what you’re doing.”
References
[1] Harford, JB, Bonifacino, JS, “Subcellular fractionation and isolation of organelles,” Curr Protocols Cell Biology, 52:3.0.1–3.0.8, 2011.