by Caitlin Smith
The purification of extracted proteins from a starting sample of cells or tissue sounds simple enough, especially when you’re talking about a total (rather than fractionated) protein sample. But in fact, finding the right total protein extraction kit for the preparation of your sample is not as simple as it seems – especially when the extracts will be used for downstream applications such as electrophoresis, immunoprecipitation, Western blotting, enzyme assays, or mass spectrometry. The kit must be capable of doing a multitude of tasks simultaneously. “The right total protein extraction kits should enable you to isolate protein from both large and small samples and from both tissues and cultured cells,” says Michael Wood, product manager for protein samples preparation at EMD Millipore. “Ideally, proteins should be rapidly isolated (less than three hours), without freeze/thaw cycles or sonication, to ensure that protein is not oxidized and maintains native conformation.”
Of course, not everyone needs proteins to maintain native conformations. But what, in fact, do you need? How important is convenience? Are additional steps worth the time? To which, if any, downstream applications will your proteins of interest be subjected? Will extraction reagents, detergents, or salts interfere with your results, or their interpretation? Considering these questions as you search for the right total protein extraction kit may help you decide what is right for you.
Ease of use
Of course, ease of use is always attractive. Especially so, it seems, to proteomics researchers, who are grappling with standards for sample preparation. “An effective proteome analysis requires the preparation of a sample to bring the wide range of protein species into the dynamic range of detection,” says Colin Heath, R&D director at G-Biosciences. “The absence of any standardized procedures for sample preparation has made proteome analysis extremely complicated, requiring a multitude of complicated skills, expensive equipment, and resources.
G-Biosciences’ FOCUS™ Proteome Kits are designed for the preparation of total protein, including soluble, insoluble, membrane, cytoplasmic, nuclear, signal, phospho- and glyco-proteins. “The FOCUS™ Proteome Kits are suitable for biological samples from tissues, cells, plants, yeast, bacteria and insects,” says Heath. “These kits are simple to use, save time, improve the quality of protein analysis and enhance the chances of discovery of novel proteins. The kits are suitable for the analysis of proteins using electrophoresis and other biochemical techniques.”
Protein Discovery offers two types of kits for protein extraction. Their UPX Universal Protein Extraction Kit is designed for unbiased extraction of both membrane and soluble proteins. Similarly, their YPX Yeast Protein Extraction Kit is targeted toward unbiased yeast protein extraction. “The simple protocol requires no mechanical disruption and works with equivalent efficiency on all yeasts regardless of morphology or growth medium,” says Andrea Mravca, VP of sales and marketing at Protein Discovery. “[Both kits] provide unbiased extraction with respect to protein size, hydrophobicity, and solubility, and are compatible with downstream workflow steps (including our Gelfree 8100 Fractionation System).” Mravca believes it is important to consider a kit’s ease-of-use, protocol length, cost, and extraction efficiency. For example, she says Protein Discovery’s UPX and YPX kits use “a short, straight-forward protocol for sample preparation in 60-90 minutes.”
Bio-Rad’s ReadyPrep kit for total protein extraction generates total protein samples in about 45 minutes that are suitable for several applications, such as isoelectric focusing and 2D electrophoresis gel separation. The kit’s extraction solution is strongly chaotropic and includes the zwitterionic detergent ASB-14. PromoKine also offers a fast and user-friendly extraction kit. Their Mammalian Whole Cell Protein Extraction Kit allows “a convenient and efficient extraction of mammalian proteins from tissues and cells under non-denaturing conditions,” says Juergen Becker, product manager for PromoKine. “The entire procedure takes less than 20 minutes, and the proteins can be used for many downstream applications such as gel electrophoresis, and Western blotting.”
Ready for downstream applications
Many downstream assays, such as enzyme assays, electrophoresis, protein folding studies, and chromatography require that extracted proteins remain active, as opposed to completely denatured, for example. “A point to consider is the application for the extracted protein,” says Heath. “Strong chaotropic denaturing extraction may result in a large pool of proteins but will not provide proteins for enzyme assays, binding studies or other applications where the functionality of a protein is important.”
EMD Millipore’s ProteoExtract Complete Mammalian Proteome Extraction Kit (C-PEK) is designed for total protein extraction from mammalian cells and tissues. “C-PEK provides a straightforward two-step isolation of complete proteomes in a single microcentrifuge tube, without the need for sonication or precipitation,” says Wood. “The procedure uses Benzonase Nuclease, a nonspecific nuclease, leading to clear, non-viscous protein solutions and improved resolution on 2D gels. Using the C-PEK kit, protein concentration is not necessary – extracted proteins are ready for immediate use in standard downstream proteomics applications.” Another choice from EMD Millipore is their CytoBuster Protein Extraction Reagent, a propriety formulation of detergents optimized for efficient extraction of soluble proteins from mammalian and insect cells. “The gentle, non-ionic composition of CytoBuster reagent enables isolation of functionally active endogenous or expressed proteins without secondary treatment such as sonication or freeze/thaw,” says Wood. “CytoBuster reagent has been specifically formulated for utilization in Western blotting, immunoprecipitation, and kinase/phosphatase assays. If phosphorylation state matters, [our] PhosphoSafe Extraction Reagent contains the same formula as CytoBuster, but also includes four phosphatase inhibitors, and is compatible with kinase assays, protein interaction analysis and other applications.”
In addition, G-Biosciences offers five different types of Protein Extraction & Lysis Buffer (PELB™) systems, for extracting active proteins from mammalian tissues or cells, bacterial cells, yeast cells and insect cells. “The PELB systems are based on a proprietary combination of organic buffering agents, mild non-ionic detergents, and a combination of various salts, to enhance extraction of soluble proteins and maintain stability of biological activities of the proteins,” says Heath. “For yeast and bacteria cells an additional LongLife lytic enzyme is also provided that requires no mixing or weighing and offers easy handling.”
Thermo Scientific Pierce offers a wide selection of protein extraction kits, including those for bacteria (Thermo Scientific B-PER Protein Extraction Reagent Kit), tissue (Thermo Scientific T-PER Protein Extraction Reagent), yeast (Thermo Scientific Y-PER Protein Extraction Reagent), insect (Thermo Scientific I-PER Protein Extraction Reagent), mammalian (Thermo Scientific M-PER Protein Extraction Reagent) and plant (Thermo Scientific P-PER Protein Extraction Reagent) cells. “Generally, except for T-PER Reagent [for tissues], one of the biggest advantages of the overall Thermo Scientific Pierce Cell Lysis Reagent product line is that they are ready-to-use, often single-component liquid reagents that don’t require mechanical cell disruption,” says Monica Noonan, technical product manager for Thermo Scientific Pierce Sample Preparation Products. “This helps preserve protein activity and maintain consistency between experiments.”
Thermo Scientific Pierce also offers several reagent tools to help make your sample more amenable to subsequent experiments. For example, their Thermo Scientific RIPA and IP Lysis Buffers are compatible with downstream applications such as reporter assays, immunoassays, and protein assays (RIPA) or immunoprecipitation (IP Lysis Buffer). Also, their Thermo Scientific Y-PER Plus Yeast Protein Extraction Reagent uses a dialyzable detergent that can be removed if it interferes with downstream applications. Another option is the Thermo Scientific Detergent Removal Resin, which can remove detergent from extracts.
G-Biosciences also offers their 2D-Xtract™ for extracting protein destined for 2D electrophoresis followed by mass spectrometry. Based on the FOCUS Extraction buffers (see below), the “2D-Xtract™ consists of optimized concentrations of urea, thiourea, CHAPS, and non-detergent sulfobetaine 201,” says Heath.
Detergents and salts: necessary contaminants
The characteristics of your proteins of interest are key when choosing an extraction kit. For the more insoluble types, the presence of stronger detergents might be required, despite being unwanted in the final sample. “The most important point to consider is the physical properties of the protein, particularly the protein’s solubility,” says Heath. “Insoluble proteins are definitely a major issue for scientists, whether native proteins (membrane proteins) or recombinant proteins that end up in inclusion bodies and require specialized lysis buffers, such as our IBS Buffer.”
“For many applications, the detergents used in the lysis buffers are very effective, but also become contaminants in the downstream analysis of the sample,” says Mravca. “Developing a cost-competitive, and effective extraction buffer that does not require a clean-up step at some point in the process would be useful.”
Though often required, strong denaturing buffers can cause unintended problems that you’ll need to watch out for. For example, the chaotropic denaturing buffer, urea, is commonly used to solubilize proteins, but as Heath explains, it entails the disadvantage of carbamylation. “Urea in water exists in equilibrium with ammonium cyanate, the level of which increases with increasing temperature and pH,” says Heath. “Cyanate reacts with á-amino and å-amino groups of proteins and induces a change in the isoelectric point of proteins. This leads to artifactual results and therefore carbamylation must be avoided.” G-Biosciences’ FOCUS Extraction Buffers were designed in part to avoid carbamylation. Supplied as a dry powder with a diluent, the FOCUS Extraction Buffers can be mixed as needed to give fresh buffers each time. “A range of buffers are available to allow researchers to optimize extraction conditions depending on the nature of their samples,” says Heath. “FOCUS Extraction Buffer-I is suitable for most applications; however for stronger solubilization effects, we recommend FOCUS Extraction Buffer-II, -III, -IV, -V or –VI and these can be used individually or sequentially for optimal extraction.”
Another hindrance in many lysis buffers is salt levels. While not a contaminant per se, salts must often be removed downstream, adding an extra step and time to the protocol. If this is an issue for your samples, you might want to consider a kit that obviates the need for this. “In most cases, there is no longer a need to desalt the samples prior to use with our Gelfree system,” says Mravca. Once you determine your downstream applications, and what that means for your sample, it should be easier to choose a total protein extraction kit from the crowd.
The image at the top of this page is from the Thermo Scientific Pierce Cell Lysis Technical Handbook .