by Caitlin Smith
Sometimes there is no better substitute for getting to your protein(s) of interest than simply breaking open the cell, heavy-handed though it may seem. However, this type of protein extraction is anything but simple. Cell lysis is a common technique used to access cellular contents. There are many kits available to help you lyse cells and use the lysate in downstream applications, such as protein purification, proteomics, X-ray crystallography, Western blotting and immunoprecipitation. Here are some considerations to guide you to the most appropriate cell lysis kit, whether your cells are bacterial, mammalian, yeast or plant.
There are several factors to consider when choosing a cell lysis kit—and choosing the best kit for your work can mean success or failure. First, consider your cell type. Not all kits are optimal for all cell types; for example, some kits are designed especially for bacterial, mammalian, plant or yeast cells. Other systems can handle multiple types, but be aware of their cell-type capabilities. Second, consider the nature and locations of the proteins you are interested in. “Is my protein localized to a particular cell compartment?” Monica Noonan, technical product manager for Thermo Scientific Pierce Sample Preparation Products, recommends considering. “And does my protein lose activity if mechanical disruption is used to lyse the cell?” Michael Wood, product manager for protein separation and preparation at EMD Millipore, recommends asking additional questions, such as “Are you looking to purify native or active recombinant proteins? Is there a requirement for any affinity purification of tagged proteins?”
One of the most important considerations includes thinking ahead to your next experiments. “Make sure that any sample preparation reagents used are compatible with not only the sample type you are using, but all other processing steps as well as downstream applications,” says Angela Crawford, product manager in protein assays at Sigma Life Science. “The goal is to use reagents that confound results as minimally as possible. This is especially important when the downstream analysis requires a functional protein, such as an activity assay.” Crawford advises researchers to look at suppliers’ claims for suitability as well as references with the same cell type or downstream applications that you plan to use.
Bacterial cells
Because we use bacteria to grow recombinant proteins, there are many choices of bacterial cell kits. For example, EMD Millipore supplies its BugBuster family of protein-extraction reagents that includes the central BugBuster Master Mix. “This combines our proprietary protein-extraction reagent with Benzonase Nuclease and rLysozyme Solution in one convenient reagent, for maximum recovery of active, soluble protein without the need for dilution or additional steps,” says Wood. “In addition, our PopCulture reagent is a detergent-based concentrate that can be added directly to cultures of E. coli to effectively extract proteins without the need for cell harvest.”
Another factor to consider is the extent of denaturing detergents that your sample can withstand. Some proteins require significant denaturing conditions to isolate them, but this can interfere with downstream applications. Other proteins are more suited to non-denaturing conditions. Sigma’s proprietary cell lysis reagents use non-denaturing zwitterionic detergents. “We test our reagents with a multitude of downstream applications, such as affinity chromatography, immunoprecipitations and Western blots, to confirm that the lysis reagents do not have any undesired effects,” says Crawford. “Our reagents are also compatible with protease inhibitors, chaotropes, salts [and] chelating and reducing agents. These compatibilities have allowed us to introduce unique products to our customers, such as our iLAP® technology. iLAP is short for integrated lysis and affinity purification. The essence of this technology is that we have combined the cell lysis and histidine-tagged recombinant-protein affinity purification in one step.” Sigma offers a CelLytic™ line that includes CelLytic B for bacteria as well as CelLytic kits for other cell types: CelLytic IB for inclusion bodies, CelLytic M for mammalian cells, CelLytic MT for tissue, CelLytic MEM for mammalian membrane extractions, CelLytic NuClear™ for mammalian nuclear extractions, CelLytic P for plant cells, CelLytic PN for plant nuclear extractions and CelLytic Y for yeast.
Mammalian cells
More mammalian cell lysis kits have become available as more researchers have started using this system. The newest cell lysis product in the Thermo Scientific Pierce portfolio is the Subcellular Protein Fractionation Kit, which helps you extract and separate cellular components. “This kit enables the stepwise separation and preparation of cytoplasmic, membrane, nuclear-soluble, chromatin-bound and cytoskeletal protein extracts from mammalian cultured cells or tissue,” says Noonan. “The extracts obtained with this kit are compatible with a variety of downstream applications and products, including Western blotting, Thermo Scientific Pierce BCA and other protein assays, the Thermo Scientific LightShift Chemiluminescent EMSA Kit and reporter-gene and enzyme-activity assays. The individual extracts generally have less than 15% contamination between fractions.” Noonan remarks that the Subcellular Protein Fractionation Kit grew from a similar product, the Thermo Scientific NE-PER Nuclear Protein Extraction Reagent. “This reagent provides efficient cell lysis and extraction of separate cytoplasmic and nuclear protein fractions in less than two hours,” she says.
EMD Millipore’s BugBuster product line also includes its Cytobuster kit, which extracts soluble proteins from mammalian cells. EMD Millipore’s more specialized lysis kits also include its “ProteoExtract Subcellular Proteome Extraction Kit (S-PEK), which is designed for fast and reproducible extraction of subcellular proteomes from mammalian cells,” says Wood.
Plant and yeast cells
It can be more difficult to extract the contents of plant cells, because they are encased in an outer cell wall. Many protocols for plant cells involve physical or mechanical disruptions (such as sonication or freeze-thaw cycles) in addition to chemical ones like detergents. “The Thermo Scientific P-PER Plant Protein Extraction Reagent is the only plant-cell lysis reagent that doesn’t require strong, mechanical disruption,” says Noonan. “Generally, one of the biggest advantages of the Thermo Scientific Pierce Cell Lysis Reagent product line is that they are ready-to-use liquid reagents that don’t require mechanical cell disruption. This helps preserve protein activity and maintain consistency between experiments.”
EMD Millipore’s Yeastbuster kit is designed to extract proteins from yeast and plants without mechanical disruption and enzymatic activity. The company also offers two reagents that support the cell lysis process, Benzonase and rLysozyme. “Benzonase Nuclease is a genetically-engineered, nonspecific endonuclease that degrades all forms of DNA and RNA (single-stranded, double-stranded, circular, linear), reducing extract viscosity and increasing protein yield. rLysozyme Solution contains a highly purified and stabilized recombinant lysozyme with specific activity 250 times greater than that of chicken egg-white lysozyme,” says Wood.
Sigma offers another kit for yeast that combines cell lysis with other activities. “The CelLytic Y Plus kit combines yeast-extraction detergents, enzymatic lysis reagents, buffers and protease inhibitors,” says Crawford. “This kit allows for purification, stabilization and detection via numerous downstream applications.”
Multiple cell-type solutions
If you work with several different cell types and need lysis kits for all, consider a system compatible with more than one type of cell. Roche’s cOmplete Lysis Kits lyse cells and protect from proteases. “They contain a mild, ready-to-use [non-denaturing] detergent that efficiently lyses mammalian, bacterial and insect cells and eliminates the need for scraping, sonication or freeze-thaw cycles,” says Oliver Seth, manager of new product marketing at Roche Applied Science. “Protein yields obtained with this kit are up to 20 to 25% higher, compared to three cycles of freeze-thaw, and approximately 20% higher than two minutes of sonication (with 50% pulse).” Included in the kits are Roche’s cOmplete Mini Protease Inhibitor Cocktail Tablets, which include both reversible and irreversible protease inhibitors. The entire cell lysis protocol takes five to 20 minutes.
For mammalian cells, bacteria and yeast, Protea Biosciences offers the ProteaPrep Cell Lysis Kit, Mass Spec Grade, for preparing samples for subsequent proteomic analysis. “This kit contains our acid-labile surfactant, which has been optimized for efficient solubilization, extraction and recovery of proteins during cell lysis,” says Haddon Goodman, product marketing manager at Protea Biosciences. “Since this kit does not contain lysozyme or harsh detergents [such as sodium dodecyl sulphate], customers are able to extract and recover cell lysates that are free of artifactual protein contaminants.” Goodman recommends that researchers understand the composition of the cell lysis buffer and kit to help with downstream analysis. “For example, we’ve designed our kit to be compatible with downstream mass spectrometry workflows (protein identification, relative quantitation, etc.), by not introducing harsh detergents or other proteins/enzymes into the kit,” he says. “The surfactants in the kit are degraded prior to mass spectrometry analysis so they do not interfere with ionization efficiency of the analytes, and no surfactant contaminating peaks are detected.”
Bio-Rad’s Cell Lysis Kit for its Bio-Plex multiplex immunoassay system (using Luminex xMap technology) also can be used for a variety of cell types. In this kit, the company has tried to balance the ability to release and isolate proteins with the ability to keep the proteins functional for downstream applications, whether they be Bio-Plex multiplex immunoassays or applications requiring more native protein confirmations. Qian Gao, Bio-Plex R&D group leader at Bio-Rad Laboratories, notes that traditionally people used to break open cells for denaturing electrophoresis gels using very strong detergents (if proteins weren’t well isolated, the gel would give erroneous molecular weights). “It is just the past few years that people have paid attention to protein interactions,” says Gao. “What if your analysis after lysis requires the protein not to be completely denatured? For example, if you are doing an immunoassay, your samples are going to interact with antibodies. If the conditions are too tough, you’re going to denature your antibody too, and then the immunoassay can’t happen. Or if you study protein-protein interactions, you need your proteins to retain their secondary conformations and some biological functions.”
To release more proteins without using more denaturing conditions (which may interfere with downstream applications, like immunoassays), Gao recommends physical treatments such as freeze-thaw methods. This combination works well with isolating nuclear proteins, which remain a challenge. He finds that freeze-thaw methods, rather than being harsh on the lysate, as others have found, instead further increases the yield of nuclear proteins. Furthermore, the effects on lysate of quick freeze-thaw (using dry ice) vs. slow freeze-thaw (using a -20°F freezer) are different—some targets release better with fast freezing, others with slow freezing.
Gao’s advice to researchers who may not be satisfied with their cell lysis result is to learn about detergents. Unfortunately, there are more than 100 different kinds, and they work differently at different concentrations. On the other hand, there are so many types of cell lysis kits available today that you are likely to find one that fits your needs.
The image at the top of this article is the Subcellular Protein Fractionation Kit from Thermo Scientific Pierce Protein Research Products.