Protein purification applications that meet your needs

Protein Purification Support | Thermo Fisher Scientific - US

protein purification applications that meet your needs

Complete solutions for FPLC (Fast Protein Liquid Chromatography) on a minimum footprint: Design your AZURA Bio purification system to your needs. Multiple Besides, the method uses column materials out of agarose or polymer material which are very We designed our systems to meet your purification challenges!. Therefore, the content of the book addresses several essential information not only Contemporary Methods in Protein Purification and Current Applications of their full chapter submissions and track important deadlines and ensure they are met. put the academic needs of scientific community first, and provide an Open. Explore this extensive selection of information to streamline your purification technical support, and field application scientists available to ensure your . Explore how to customize an NGC System to meet your specific purification needs. ChromLab Software makes purifying proteins on the NGC Chromatography.

A systematic approach can be used to develop a purification strategy.

protein purification applications that meet your needs

Purification challenges Successful protein purification requires following a multi-step approach to perform Capture, intermediate Purification and Polishing CiPP. Specific objectives are assigned to each: These cover all major chromatographic techniques and steps.

This allows you to: Choose logical combinations of purification techniques based on the main benefits of the technique and the condition of the sample at the beginning and end of each step Minimize sample handling between purification steps by combining the best techniques Increase yield while saving both time and money by using as few steps as possible Purification of non-tagged proteins 23 24 Rapid three-step purification of a labile, oxygen-sensitive enzyme Fig.

LMW Purification of non-tagged proteins Conclusions Rapid purification method with modern, preparative separation media and strategic purification design Isolation of 1 mg of an active, highly-labile enzyme to crystallization grade purity Timesavings of purification method: Capture and intermediate purification: Rainbow trout vitellogenin Fig. Rainbow trout vitellogenin CH-Vtg: Immunoblotting was performed with BN-5 anti-salmon Vtg antibody; complexes were immunodetected by enhanced chemiluminescent method after transfer of proteins to a nitrocellulose paper.

Conclusions Vitellogenin Vtg was purified from three estradiol-treated teleost fish: Logical combinations of chromatographic steps is shown in Fig.

A guide to the suitability of each technique for the stages of the Capture, intermediate Purification and Polishing CiPP strategy is shown in Table 13B.

If nothing is known about the target proteins, an effective general approach is: To identify the products that best suit your needs, please consult our selection guides, available at: Hydrophobic Interaction chromatography, RPC: Reversed phase chromatography Automated chromatography system s cited System Quantity Code no. Proteins are expressed either in cytoplasm or secreted. Over-expression of proteins in some cases leads to accumulation of the proteins in insoluble polypeptide aggregates, called inclusion bodies.

Purification challenges To transform the proteins insoluble in within the inclusion bodies into a useful soluble, bioactive protein. Solutions Chromatographic on-column refolding offers: Automated procedures Reliable, simple scale-up Refolding and purification in a single step, simultaneously High compatibility and chemical stability with different additives, reducing agents, denaturing agents and detergents Convenient optimization of separation conditions using pre-programmed method templates and scouting functions Refolding proteins from inclusion bodies 27 28 One-step on-column refolding and purification of a Histidine-tagged protein from E.

On-column refolding and purification Solubilizing buffer: Histidine-tagged, solubilized single chain Fv antibody fragment Fab 57P, 1 ml conc. Sensogram of the interaction between immobilized peptide and refolded protein System: Biacore system 2 Sensor surface: Immobilized peptide C16V 37 tobacco mosaic virus with affinity to scfv57p Blank surface: Immobilized with peptide with no affinity to scfv57p Yield: Example of a dual-gradient ion-exchange refolding procedure.

Protein Purification Systems

A 28 Urea M. Chemical conditions also vary from protein to protein. Several experiments are required to identify the optimal refolding process. Ion Exchange chromatography; GF: Polyethylene glycol Reprinted with the kind permission of Elsevier 29 30 On-column refolding products Quantity Code no. For proteome analysis of human serum, many researchers are studying low abundance proteins.

One of their primary goals is to find useful biomarkers for different diseases and conditions. Mass spectrometry MS analysis is a very important tool for protein analysis.

Certain molecules degrade proteins in cell culture supernatants, bacterial lysate or serum. Challenges High abundance proteins, such as albumin and immunoglobulins, make it difficult to detect low abundance proteins. Proteases included in human plasma can damage the sample if not removed. MS analysis can not be performed when certain buffer salts are present in the solution. M NaCl, ph 7. Removal of trypsin-like serine proteases Conclusions Proteases included in human plasma can damage the sample if not removed 1 ml human plasma filtered through a.

HiTrap Desalting, 1 5 ml, 3 5 ml, 5 5 ml Sample vol.: HiTrap Desalting 1X5 ml in series Fig. HiTrap Desalting 3X5 ml in series Fig. HiTrap Desalting 5X5 ml in series. Connect HiTrap Desalting columns in series for fast and simple scale-up. Desalting using cross-flow filtration for ml sample volumes Fig Ideal for processing ml of feedstock Reusable filters deliver reproducible results Sample preparation 33 34 Removal of high abundance proteins Prepacked columns Quantity Code no.

GLP Automatic method development and optimization Automatic buffer preparation Automatic ph scouting Automatic media or column scouting Automatic multistep purification Method development and scale-up Sanitary design cgmp Scale-up, process development, and transfer to production Fully-automated, high-throughput, unattended operations UNICORN control system: It offers easy-to-use, editable, method wizards for all major applications and techniques.

protein purification applications that meet your needs

Together with our sales and applications specialists they engage in close collaborations with customers and other global leaders within the life science, biotech and biopharmaceutical industries.

With a 5 year proven track record of pioneering and improving most of the techniques used in protein purification, we remain committed to making your applications faster, simpler more reliable and productive.

Two recent examples include: This latter combination offers one-step simplicity and up to four times higher binding capacity for purfying histidine-tagged proteins. Our media and columns are produced according to validated methods and are strictly quality control-tested.

This ensures high batch-tobatch reproducibility of our media and prepacked columns while helping you achieve reproducible results. All our products are manufactured and delivered in accordance to ISO 91 and are backed up with technical data.

GE Healthcare. Protein purification Applications that meet your needs

Critical materials used to construct equipment i. We offer full tracability of all material sources. Plus, we invest in media characterization technologies that prove our media meet their specifications and thereby offer you total reassurance. In fact, every minute of every working day they help customers solve purification challenges. Our sales professionals are always available to assist you.

You can contact our technical support scientists either online or over the phone. Our distribution professionals as well as our inventory management systems and procedures ensure timely deliveries. Our service professionals provide standard and customized agreements to support equipment and process uptime. Scientific forums We establish environments for professionals to share experience and knowledge. Online, we offer education centers and users clubs.

We hold or are active in thousands of face-to-face discussion forums every year.

Protein Purification

Courses and training Boosting the knowledge and efficiency levels of your teams offers you a lasting competitive advantage. We offer both general and customized training to address your specific needs.

On average, over 45 engineers and scientists attend our Fast Trak courses every year. Technical support literature Many university departments around the world use our technique handbooks as educational supplements.

We support you with technical literature, including product catalogs, scientific posters, application notes and more. These are regularly updated to reflect current developments and are available at: This mechanism works best for chromatographic procedures based on a very specific interaction i.

Batchwise elution does not offer any resolution, but it is ideal for getting rid of contaminants very quickly. It requires prior knowledge of buffer conditions required for displacement of the protein of interest. Stepwise - multiple batchwise elutions are performed sequentially, with more stringent conditions in each step. In stepwise elution, the number of fractions collected is dependent upon the number of sequential elution conditions. Stepwise elution provides better resolution than batchwise elution, but poorer resolution than linear gradient elution.

Linear Gradient - multiple consecutive fractions are collected while elution conditions are adjusted in a linear fashion. A linear gradient offers the highest resolution for ion exchange chromatography and hydrophobic interaction chromatography. Typically, a large number of consecutive fractions are collected. Size exclusion chromatography does not require any of these elution methods, as no interactions occur between the protein and the stationary phase. Protein is loaded onto the column, and a large number of fractions are collected until all proteins are eluted, without altering buffer conditions throughout the procedure.

Ideally, the elution buffer of a column is compatible with the subsequent column, eliminating the need for buffer exchange or dialysis between purification steps. Less common chromatography methods Hydroxyapatite hydroxylated calcium phosphate is a protein purification technique originally described in the mid s [ 42 ]. The commercial availability of spherical hydroxyapatite has made hydroxyapatite column chromatography an accessible technique [ 43 ].

The mechanisms underlying the interaction of proteins with hydroxyapatite columns is complex. Proteins are most commonly eluted with phosphate gradients.

Hydroxyapatite chromatography has been used successfully in the purification of therapeutic grade antibodies [ 44 ]. Chromatofocusing is a column chromatographic method that separates proteins on the basis of their pI. Proteins are bound to specialised ion-exchange media and eluted with a pH gradient. Analysis of Fraction Purity After each chromatographic separation, fractions must be analyzed to determine the fractions that contain the protein of interest and the relative purity of each of those fractions.

This analysis is necessary after each step to decide which fractions should be pooled for subsequent use. To determine purity, an assay is required that can measure the amount of a specific protein relative to the amount of total protein. The following assays are routinely used for purity analysis: Commercially available stains allow for a visual representation of all proteins in the sample, offering a qualitative assessment of protein purity Figure SDS-PAGE is not ideal for high-throughput analysis of fractions and can take several hours; however, it is most often used because it is easy, inexpensive, and suitable for any protein.

Protein Purification Systems - GE Healthcare Life Sciences

Gel is stained for the visualization of all proteins. Spectroscopy - a method for analyzing optical properties of proteins. This technique for analyzing protein purity is only suited for proteins, such as cytochrome Ps, that have a unique spectroscopic feature.

Proteins in this family absorb light at a wavelength where other proteins do not around nanometers [ 46 ]so a comparison of absorbance at nanometers versus nanometers the wavelength at which all proteins absorbcan provide a quantitative measure of protein purity. This method is fast and high-throughput, but only suitable for some proteins. Protein Activity - an enzymatic test that depends on the protein of interest.

This method of assessing protein purity is often coupled with another form of protein concentration determination to calculate activity relative to total protein concentration.

Activity assays are only suitable for proteins with activity than can easily be monitored in a high-throughput format, such as proteases. For some proteins, activity assays provide a fast and reliable method for protein detection. Activity measurement is often ideal for enzymes, because protein that has lost activity can be excluded from subsequent use.

Purified Protein Storage When proteins are deemed pure enough for use in experimental studies, they should be stored appropriately. The selection of a final storage buffer is just as important as the selection of buffers used during the purification scheme, and should depend on stability of the protein and conditions required for the downstream application of the purified protein.

Oftentimes, size exclusion chromatography is selected as a final step in the purification scheme, as the storage buffer can be used in this chromatographic step to effectively exchange the buffer.

The pure fractions can be pooled for immediate storage. Alternatively, the final pooled fractions can be dialyzed into the selected buffer before storage. Protein storage conditions depend on the protein of interest, and should be optimized so the protein maintains structural and functional stability over long periods of storage. Additives are often included in the storage buffer to enhance the lifetime of purified proteins under storage conditions, and trial and error is often required to determine optimum conditions, as every protein behaves differently.

Thus, there is great interest in solving structure of membrane proteins. A crucial step in the purification of integral membrane proteins is their solubilisation from the lipid bilayer whilst retaining their functional integrity. The typical approach involves the isolation of intracellular membranes by centrifugation followed by detergent solubilisation of integral membrane proteins and high speed centrifugation to remove insoluble membrane residues [ 50 - 52 ].

A wide range of detergents have been employed for membrane protein solubilisation [ 53 - 55 ] and, in the absence of literature or laboratory precedent, the investigator will need to empirically determine the best detergent for their particular protein.

The solubilised membrane protein may then be purified by column chromatography in essentially the same way as for soluble proteins.