Together in Protein Research

Your curiosity and engagement is the foundation for uncovering new pathways and cures In protein research, going from idea to pure characterized protein requires many steps and techniques. Understanding the principles behind different tools uses up time that could be spent generating data and progressing research. Don’t leave success to chance. Use our guide to get active and pure proteins. Introduction Analysis Purification Sample prepSample prep 026 Environmental testing for water, air, and soil Sample prep. Throughout sample preparation, protein purification, and analysis — protein damage needs to be avoided to ensure that activity is maintained. Some proteins are more sensitive than others. Understanding your protein’s properties helps determine which conditions to use and avoid. Some proteins, like secreted proteins, have low expression levels. When expression levels are low, it becomes even more important to maintain a high yield in each step. Sample preparation depends on protein type. Membrane proteins, protein complexes, and secreted proteins have different sample preparation protocols. For intracellular proteins, scientists lyse cells to release the target protein — this can be done through sonication, homogenization, freezing and thawing, or chemical agents. For extracellular proteins obtained from mammalian or insect cells, cell harvest can be directly applied. Scientists often perform cell harvest using centrifugation. In most applications the supernatant is transferred to protein purification containing the target protein or it is analyzed directly to perform identification and determine activity. If needed, filtration can be used to remove particles after centrifugation, or before chromatography to protect your columns. Desalting at laboratory scale is a well-proven, simple, and fast method that removes low molecular weight impurities and transfers the sample into required buffer in a single step. Desalting or buffer exchange can be used prior to a chromatography step or between purification steps for sample conditioning. What determines a successful purification scheme? The output of a protein purification scheme is traditionally described in terms of purity, homogeneity, and yield. Optimization of one of the output parameters can usually only be achieved at the expense of the other output parameters, and each purification step will therefore be a compromise. Example of output parameters Purity measures: • Electrophoresis, specified as target protein-to-total protein ratio. • Size exclusion chromatography. Homogeneity measures: • Size exclusion chromatography, specified as monomer-toaggregate content. • Native electrophoresis. Yield measures: • Absorption at 280 nm and using the extinction coefficient for the target protein. • Activity assay, specified as U/L. • Concentration using surface plasmon resonance or immunebased assays. Purity Homogeneity YieldWhat phases constitute a chromatographic step? Each chromatographic step can be split into several phases, such as sample load, wash, and elution. Each phase involves different factors (such as pH, conductivity, additives, and flow rate). All have a significant effect on the outcome of the chromatographic step. What chromatography methods can I choose from? Chromatographic methods depending on the properties of your target protein and the properties of other substances in the sample. 9 How do I select a chromatography resin? Purification efficiency is dependent on the chromatography resin selected for each technique. The particle size of the resin strongly affects efficiency and flow resistance. A resin with large beads gives chromatography columns with low resolution (broad peaks), but generates low backpressure. Small beads give higher resolution (narrow peaks), but generate higher backpressure. A general principle of choosing chromatography resins is a larger bead size for early purification steps, and smaller bead size for later steps, where demand on purity is increased. Protein property Method Specific ligand recognition Affinity chromatography (AC) Metal ion binding Immobilized metal ion affinity chromatography (IMAC) Charge Ion exchange chromatography (IEX) Size Size exclusion chromatography (SEC) Hydrophobicity Hydrophobic interaction chromatography (HIC) Reversed phase chromatography (RPC) Combination of several protein properties, such as charge, size or hydrophobicity Multimodal chromatography (MM) What are the steps in a protein purification protocol? CiPP (capture, intermediate purification, polishing) purification strategy can be used to structure your purification protocol: • The initial capture stage isolates, concentrates, and stabilizes your protein. • Intermediate purification removes bulk contaminants. • The final polishing step removes the most difficult impurities, such as aggregates of the target protein. In a research setting, purity requirements are often less important than yield and you may therefore only need a chromatography capture step to achieve your goal. A general rule is the higher protein purity you need, the more purification steps you will have to use in your workflow. However, additional chromatography steps increase purity, but decrease yield of active protein.