Magnetic separation technology, using magnetic particles, is quick and easy method for sensitive and reliable capture of specific proteins, genetic material and other biomolecules. The technique offers an advantage in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap and often highly scalable. Moreover, techniques employing magnetism are more amenable to automation and miniaturization. Now that the human genome is sequenced and about 30,000 genes are annotated, the next step is to identify the function of these individual genes, carrying out genotyping studies for allelic variation and SNP analysis, ultimately leading to identification of novel drug targets. In this post-genomic era, technologies based on magnetic separation are becoming an integral part of todays biology laboratory. This article briefly reviews the selected applications of magnetic separation techniques in the field of biotechnology, biomedicine and drug discovery.
Experimental and clinical studies often require highly purified cell populations. FACS is a technique of choice to purify cell populations of known phenotype. Other bulk methods of purification include panning, complement depletion and magnetic bead separation. However, FACS has several advantages over other available methods. FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density. In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker. FACS allows the purification of individual cells based on size, granularity and fluorescence. In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1). Negative selection of unstained cells is also possible. FACS purification requires a flow cytometer with sorting capacity and the appropriate software. For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser. The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells. The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2). The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb. Sorting parameters can be adjusted depending on the requirement of purity and yield. Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.
We present a new method for rapid purification to near homogeneity of sequence specific DNA binding proteins based on magnetic separation. The method is described for the purification of the yeast transcription factor tau. DNA affinity Dynabeads (monodisperse superparamagnetic particles) specifically bind the protein in the presence of competitor DNA. By magnetic separation, wash and elution, highly enriched transcription factor preparations are obtained within minutes. In less than an hour with three cycles of adsorption, nearly homogeneous factor tau was obtained. The factor preparation contained mainly two polypeptides of 100 and 140 kDa and was fully active in transcription and DNA binding assays. This procedure should work for any high-affinity sequence-specific DNA binding protein with only minor modifications.
Glycosylation is the most common form of posttranslational modification of proteins (50–80%). The isolation, discovery, and subsequent identification of glycosylated peptides and proteins is becoming more and more important in glycoproteomics and diagnosis. MALDI-TOF mass spectrometry is an ideal technique for identifying peptides and proteins and their corresponding modifications. The enrichment of glycosylated peptides and proteins from different sources can be attained by affinity chromatography supported by functionalized magnetic particles. Covalent coating of magnetic beads with Concanavalin A (ConA) and diboronic acid was performed by carbodiimide and poly-glutaraldehyde methods, respectively. The functionalized beads were employed to establish and optimize protocols for the binding and detection of glycosylated peptides and proteins with respect to an automated workflow and the subsequent detection and identification by MALDI-TOF mass spectrometry. For several model proteins, the capture and identification could be demonstrated by SDS-PAGE and MALDI-TOF mass spectrometry. According to the type of glycosylation (high man-nose, hybrid, or complex type) the different proteins were enriched by ConA or boronic acid–functionalized beads.
Glycosylation; Concanavalin A; boronic acid; magnetic particles; MALDI-TOF MS
Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated LPS contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures.
plasmid DNA purification; non-ionic detergents; Hofmeister salts
Magnetic isolation is a promising method for separating and concentrating pancreatic islets of Langerhans for transplantation in Type 1 Diabetes patients. We are developing a continuous magnetic islet sorter to overcome the restrictions of current purification methods that result in limited yield and viability. In Quadrupole Magnetic Sorting (QMS) islets are magnetized by infusing superparamagnetic microbeads into islets’ vasculature via arteries that serve the pancreas. The performance of the islet sorter depends on the resulting speed of the islets in an applied magnetic field, a property known as magnetophoretic mobility. Essential to the design and successful operation of the QMS is a method to measure the magnetophoretic mobilities of magnetically infused islets. We have adapted a Magnetic Particle Tracking Velocimeter (MPTV) to measure the magnetophoretic mobility of particles up to 1000 microns in diameter. Velocity measurements are performed in a well-characterized uniform magnetic energy gradient using video imaging followed by analysis of the video images with a computer algorithm that produces a histogram of absolute mobilities. MPTV was validated using magnetic agarose beads serving as islet surrogates and subjecting them to QMS. Mobility distributions of labeled porcine islets indicated that magnetized islets have sufficient mobility to be captured by the proposed sorting method, with this result confirmed in test isolations of magnetized islets.
Particle tracking velocimetry; magnetic flow sorter; pancreatic islets isolation; magnetic particles
The growing importance of mass spectrometry for the identification and characterization of bacterial protein toxins is a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, especially when these techniques are combined with affinity methods. Here we describe a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization-time of flight mass spectrometry for selective purification and detection of staphylococcal enterotoxin B (SEB). SEB is a potent bacterial protein toxin responsible for food poisoning, as well as a potential biological warfare agent. Unambiguous detection of SEB at low-nanogram levels in complex matrices is thus an important objective. In this work, an affinity molecular probe was prepared by immobilizing anti-SEB antibody on the surface of para-toluene-sulfonyl-functionalized monodisperse magnetic particles and used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB immunoglobulin G and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB could be detected directly “on beads” by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, “off beads” after its acidic elution. Application of this method to complex biological matrices was demonstrated by selective detection of SEB present in different matrices, such as cultivation media of Staphylococcus aureus strains and raw milk samples.
Functional protein analysis often calls for lengthy, laborious in vivo protein expression and purification, and can be complicated by the lack of stability of the purified protein. In this study, we demonstrate the feasibility of a simplified procedure for functional protein analysis on magnetic particles using cell-free protein synthesis of the catalytic subunit of human cAMP-dependent protein kinase as a HaloTag® fusion protein. The cell-free protein synthesis systems provide quick access to the protein of interest, while the HaloTag technology provides efficient, covalent protein immobilization of the fusion protein, eliminating the need for further protein purification and minimizing storage-related stability issues. The immobilized cPKA fusion protein is assayed directly on magnetic beads and can be used in inhibitor analyses. The combination of rapid protein synthesis and capture technologies can greatly facilitate the process of protein expression and activity screening, and therefore, can become a valuable tool for functional proteomics studies.
cell-free expression; in vitro translation; HaloTag; protein immobilization; magnetic particles; PKA; kinase
Recombinant protein expression and purification remains a fundamental issue for biotechnology. Recently we found that two short self-assembling amphipathic peptides 18A (EWLKAFYEKVLEKLKELF) and ELK16 (LELELKLKLELELKLK) can induce the formation of active protein aggregates in Escherichia coli (E. coli), in which the target proteins retain high enzymatic activities. Here we further explore this finding to develop a novel, facile, matrix-free protein expression and purification approach.
In this paper, we describe a streamlined protein expression and purification approach by using cleavable self-aggregating tags comprising of one amphipathic peptide (18A or ELK16) and an intein molecule. In such a scheme, a target protein is first expressed as active protein aggregate, separated by simple centrifugation, and then released into solution by intein-mediated cleavage. Three target proteins including lipase A, amadoriase II and β-xylosidase were used to demonstrate the feasibility of this approach. All the target proteins released after cleavage were highly active and pure (over 90% in the case of intein-ELK16 fusions). The yields were in the range of 1.6-10.4 μg/mg wet cell pellet at small laboratory scale, which is comparable with the typical yields from the classical his-tag purification, the IMPACT-CN system (New England Biolabs, Beverly, MA), and the ELP tag purification scheme.
This tested single step purification is capable of producing proteins with high quantity and purity. It can greatly reduce the cost and time, and thus provides application potentials for both industrial scale up and laboratorial usage.
Quadrupole magnetic field-flow fractionation is a relatively new technique for the separation and characterization of magnetic nanoparticles. Magnetic nanoparticles are often of composite nature having a magnetic component, which may be a very finely divided material, and a polymeric or other material coating that incorporates this magnetic material and stabilizes the particles in suspension. There may be other components such as antibodies on the surface for specific binding to biological cells, or chemotherapeutic drugs for magnetic drug delivery. Magnetic field-flow fractionation (MgFFF) has the potential for determining the distribution of the magnetic material among the particles in a given sample. MgFFF differs from most other forms of field-flow fractionation in that the magnetic field that brings about particle separation induces magnetic dipole moments in the nanoparticles, and these potentially can interact with one another and perturb the separation. This aspect is examined in the present work. Samples of magnetic nanoparticles were analysed under different experimental conditions to determine the sensitivity of the method to variation of conditions. The results are shown to be consistent and insensitive to conditions, although magnetite content appeared to be somewhat higher than expected.
magnetic nanoparticles; field-flow fractionation; characterization; quadrupole magnet; magnetic field-flow fractionation; magnetic dipole interaction
We have developed a range of magnetic beads for protein and/or peptide sample isolation and fractionation that can be utilised in proteomics strategies and workflows.
A significant challenge in proteomics today is overcoming the dynamic range of protein abundance. High throughput proteomics requires efficient methods to analyze complex protein mixtures. It is often necessary to reduce sample complexity for many proteomics strategies. The use of magnetic beads for sample preparation enables protocols to be automated and throughput to be increased. The kinetics of bead-based sample preparation is very efficient and washing can be done thoroughly. In addition, preparation is flexible regarding sample and buffer volumes. Consequently, low abundant proteins can be concentrated from large sample volumes.
We have developed protein purification methods based on the use of magnetic bead technology. These methods are used for the fractionation of complex protein samples and for isolation of proteins or peptides.
The generation of peptide mass fingerprints followed by a database search is a common tool for the mass spectrometric identification of proteins. To provide a high sensitivity, proteins must be efficiently purified and concentrated prior to enzymatic digestion. Common desalting procedures, like ultrafiltration or dialysis, are very time consuming and work best for high protein concentrations. Another critical point is the proteolysis of the investigated protein, which works efficiently only with concentrated protein solutions. Therefore, efficient concentration and simultaneous purification using solid phase extraction (SPE) will be the method of choice to receive pure and highly concentrated protein solutions prior to enzymatic digestion. In this work, we manufactured magnetic reversed phase particles for the efficient purification and simultaneous concentration of protein samples with volumes up to several millilitres. The SPE procedure was compared with dialysis using commercial available microconcentrators with a cut-off membrane. Due to the magnetic core, each washing and elution step could be performed within 15 minutes. Then, the bound protein was digested directly on the beads, resulting in a remarkable increase of protein detection and better mass structural analysis. Useful MOWSE scores were achieved using bovine serum albumin as a model protein with concentrations as low as 50 ng/ml (720 pM). Compared to the dialysis procedure, which needs several hours, the isolation and purification of protein can be performed in minutes with the reversed phase particles.
Magnetic Split-flow thin (SPLITT) fractionation is a newly developed technique for separating magnetically susceptible particles. Particles with different field-induced velocities can be separated into two fractions by adjusting applied magnetic forces and flow-rates at inlets and outlets.
Magnetic particles, Dynabeads, were used to test this new approach of field-induced velocity for susceptibility determination using magnetic SF at different magnetic field intensities. Reference measurements of magnetic susceptibility were made using a superconducting quantum interference device (SQUID) magnetometer. Various ion-labeled red blood cells (RBC) were used to study susceptibility determination and throughput parameters for analytical and preparative applications of magnetic SPLITT fractionation (SF), respectively. Throughputs were studied at different sample concentrations, magnetic field intensities, and channel flow-rates.
The susceptibilities of Dynabeads determined by SPLITT fractionation (SF) were consistent with those of reference measurement using a superconducting quantum interference device (SQUID) magnetometer. Determined susceptibilities of ion-labeled RBC were consistent within 9.6% variations at two magnetic intensities and different flow-rates. The determined susceptibilities differed by 10% from referenced measurements. The minimum difference in magnetic susceptibility required for complete separation was about 5.0 × 10-6 [cgs]. Sample recoveries were higher than 92%. The throughput of magnetic SF was approximately 1.8 g/h using our experimental setup.
Magnetic SF can provide simple and economical determination of particle susceptibility. This technique also has great potential for cell separation and related analysis. Continuous separations of ion-labeled RBC using magnetic SF were successful over 4 hours. The throughput was increased by 18 folds versus early study. Sample recoveries were 93.1 ± 1.8% in triplicate experiments.
Alkyl hydroperoxide reductase (AhpC) of Helicobacter pylori is considered as a diagnostic antigen. Therefore, this antigen can be used to detect H. pylori infection by stool immunoassays such as ELISA. The aim of this study was to simplify the AhpC protein purification procedures.
For whole cell protein extraction, the bacterial cells were ruptured by octly-β-D glucopyranoside. The isolation and purification of AhpC protein were attempted by various techniques including ammonium sulfate precipitation, dialysis, preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution.
A simple method was used for protein purification AhpC protein. One-dimensional preparative gel electrophoresis allows a single and short purification step; the high resolution capacity of this technique leads to a high level of purity of the protein. Moreover, it avoids contamination by other non-specific proteins which often appear during protein purification by column chromatography.
The present method is simple, rapid and makes it possible to preparate AhpC from H. pylori.
Alkyl hydroperoxide reductase; AhpC; Electroelution; Helicobacter pylori; SDS-PAGE
Histidine-rich peptides are commonly used in recombinant protein production as purification tags, allowing the one-step affinity separation of the His-tagged proteins from the extracellular media or cell extracts. Genetic engineering makes feasible the post-purification His-tag removal by inserting, between the tag and the main protein body, a target site for trans-acting proteases or a self-proteolytic peptide with regulatable activities. However, for technical ease, His tags are often not removed and the fusion proteins eventually used in this form. In this commentary, we revise the powerful biological properties of histidine-rich peptides as endosomolytic agents and as architectonic tags in nanoparticle formation, for which they are exploited in drug delivery and other nanomedical applications. These activities, generally unknown to biotechnologists, can unwillingly modulate the functionality and biotechnological performance of recombinant proteins in which they remain trivially attached.
The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 Å, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.
Elastin provides recoil to tissues subjected to repeated stretch, such as blood vessels and the lung. It is encoded by a single gene in mammals and is secreted as a 60–70 kDa monomer call tropoelastin. The functional form of the protein is that of a large, highly crosslinked polymer that organizes as sheets or fibers in the extracellular matrix. Purification of mature, crosslinked elastin is problematic because its insolubility precludes its isolation using standard wet-chemistry techniques. Instead, relatively harsh experimental approaches designed to remove non-elastin ‘contaminates’ are employed to generate an insoluble product that has the amino acid composition expected of elastin. Although soluble, tropoelastin also presents problems for isolation and purification. The protein’s extreme stickiness and susceptibility to proteolysis requires careful attention during purification and in tropoelastin-based assays. This article describes the most common approaches for purification of insoluble elastin and tropoelastin. It also addresses key aspects of studying tropoelastin production in cultured cells, where elastin expression is highly dependent upon cell type, culture conditions, and passage number.
elastin; elastic fiber; tropoelastin; microfibrils; fibrillin; purification
We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic-bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers.
Some biotechnological inventions involve expensive, sophisticated machines. Others are relatively simple innovations that nevertheless address, and solve difficult problems. Synthesis and purification of highly hydrophobic peptides can be a difficult and challenging task, particularly when these peptides have low solubility in both aqueous and organic solvents. Here we describe the synthesis and purification of a series of peptides derived from the hydrophobic C-terminus of the 42-residue form of amyloid β-protein (Aβ42), a peptide believed to be the primary cause for Alzheimer’s disease (AD). The series of C-terminal fragments (CTFs) had the general formula Aβ(x-42), x=28–39, which potentially can be used as inhibitors of Aβ42 assembly and neurotoxicity. Synthesis and purification of peptides containing 8-residues or less were straightforward. However, HPLC purification of longer peptides was problematic and provided <1% yield in particularly difficult cases due to very poor solubility in the solvent systems used both in reverse- and in normal phase chromatography. Modification of the purification protocol using water precipitation followed by removal of scavengers by washing with diethyl ether circumvented the need for HPLC purification and provided these peptides with purity as high as HPLC-purified peptides and substantially increased yield.
Obtaining highly purified proteins is essential to begin investigating their functional and structural properties. The steps that are typically involved in purifying proteins can include an initial capture, intermediate purification, and a final polishing step. Completing these steps can take several days and require frequent attention to ensure success. Our goal was to design automated protocols that will allow the purification of proteins with minimal operator intervention. Separate methods have been produced and tested that automate the sample loading, column washing, sample elution and peak collection steps for ion-exchange, metal affinity, hydrophobic interaction and gel filtration chromatography. These individual methods are designed to be coupled and run sequentially in any order to achieve a flexible and fully automated protein purification protocol.
protein purification; aspartate semialdehyde dehydrogenase; ÄKTA Explorer; automated purification protocols
Fluorescent dyes provide specific, sensitive, and multiplexed detection of nucleic acids. To maximize sensitivity, fluorescently labeled reaction products (e.g., cycle sequencing or primer extension products) must be purified away from residual dye-labeled precursors. Successful high-throughput analyses require that this purification be reliable, rapid, and amenable to automation. Common methods for purifying reaction products involve several steps and require processes that are not easily automated. Prolinx®, Inc. has developed RapXtract™ superparamagnetic separation technology, affording rapid and easy-to-perform methods that yield high-quality product and are easily automated. The technology uses superparamagnetic particles that specifically remove unincorporated dye-labeled precursors. These particles are efficiently pelleted in the presence of a magnetic field, making them ideal for purification because of the rapid separations that they allow. RapXtract-purified sequencing reactions yield data with good signal and high Phred quality scores, and they work with various sequencing dye chemistries, including BigDye and near-infrared fluorescence IRDyes. RapXtract technology can also be used to purify dye primer sequencing reactions, primer extension reactions for genotyping analysis, and nucleic acid labeling reactions for microarray hybridization. The ease of use and versatility of RapXtract technology makes it a good choice for manual or automated purification of fluorescently labeled nucleic acids.
; paramagnetic particles; fluorescent dye labels; automation; removal of unincorporated dye-labeled nucleotides
The avian myeloblastosis virus pp19 protein was separated from the other virus proteins by a rapid and simple purification procedure which yields milligram amounts of homogeneous protein. This protein was then fragmented by digestion with cyanogen bromide. When the mixture of the cyanogen bromide peptides was passed through a 60S avian myeloblastosis virus RNA-cellulose column, only one peptide bound with high affinity to the resin. The peptide migrated on a sodium dodecyl sulfate-polyacrylamide gel with an approximate molecular weight of 2,900 and will be referred to as the p3B peptide. This peptide was also isolated directly by chromatography of the cyanogen bromide-digested pp19 protein on a reverse-phase high-pressure liquid chromatography column. It was again the only cyanogen bromide peptide of the pp19 protein that bound to the RNA affinity resin. The p3B peptide is a basic peptide, as was seen by its rapid migration on acid-urea-polyacrylamide gels and its amino acid composition. A partial amino acid sequence analysis of the p3B peptide indicated that it was derived from the amino terminus of the intact protein. Although the p3B peptide bound to 60S RNA, it did not demonstrate the selective binding of native pp19 to regions of the RNA containing secondary structure.
In the Quadrupole Magnetic Sorter (QMS) magnetic particles enter a vertical flow annulus and are separated from non-magnetic particles by radial deflection into an outer annulus where the purified magnetic particles are collected via a flow splitter. The purity of magnetically isolated particles in QMS is affected by the migration of nonmagnetic particles across transport lamina in the annular flow channel. Computational Fluid Dynamics (CFD) simulations were used to predict the flow patterns, pressure drop and nonspecific crossover in QMS flow channel for the isolation of pancreatic islets of Langerhans. Simulation results were compared with the experimental results to validate the CFD model. Results of the simulations were used to show that one design gives up to 10% less nonspecific crossover than another and this model can be used to optimise the flow channel design to achieve maximum purity of magnetic particles.
Quadrupole Magnetic Sorter; Computational Fluid Dynamics; Annulus Flow; Nonspecific Crossover
Affinity purification is a powerful tool for protein enrichment in proteomics studies. We here present a fully automated system for purification of His-tag proteins and IgG using Ni2+/Co2+ and Protein A magnetic beads, respectively. Reagents for His-tag protein or IgG purification are pre-dispensed in a sealed cartridge for automated runs on a Magtration 12GC robot. The automated purification is based on Magtration technology to perform magnetic bead separation similar to a filtration process in a pipette tip. An optimized protocol has been developed for the automated protein purification. High protein purity and yields were obtained using this automated system. His-tag protein human galectin-1 was purified to approx. 1.6 mg with 12 samples processed in parallel within 30 min on the 12GC robot. This system was also used to screen the expression of His-tag water-soluble proteins and inclusion bodies in bacterial cells, even at a very low expression level. Using Protein A magnetic beads and corresponding pre-filled reagent cartridges, various amounts of human serum (15–80 μL) and the magnetic beads (100–200 mg) were tested on the robotic system. With 30 μL serum and 150 mg magnetic beads, we purified IgG with a high yield of 230 μg. A total of approx. 2.8 mg IgG can be obtained within 60 min with 12 samples run in parallel on the robot. The magnetic beads after the affinity purification can be regenerated by automation for repeated use. Magtration robotic system can be extended for purification of GST-tag fusion proteins and Immunoprecipitation by automation. We have provided an automated protein purification system with a Magtration robot and pre-filled reagent cartridges for rapid and multiparallel processing of different proteins.
Malaria parasites generate free haem upon catabolism of host haemoglobin during their intraerythrocytic growth cycle. In order to minimize oxidative toxicity of the ferric iron, the free haem molecules are polymerized into the biomineral beta-haematin (commonly referred to as haemozoin). Haemozoin crystals are paramagnetic, and this property can be exploited for the purification of late stage parasites as they contain larger haemozoin crystals than early stage parasites and uninfected cells. Commercially available magnets that were originally developed for the purpose of antibody-mediated cell purification are widely used for this purpose. As these methods are not necessarily optimized for parasite purification, the relationship between magnetic field strength and the quantity and quality of yield during parasite purification was explored.
Inexpensive rare-earth neodymium magnets with commercially available disposable columns were employed to explore the relationship between magnetic field strength and recovery of free haemozoin and infected erythrocytes (iRBCs).
Yields of free haemozoin increased nearly linearly with increasing magnetic field strength to the strongest fields tested (8,500 Gauss). Stronger magnetic fields also improved the recovery of iRBCs with no detrimental effects on parasite viability. An in-house constructed magnetic stand, built for $75 in materials, produced superior results when compared with much more expensive commercial products.
Existing protocols for the magnetic purification of free haemozoin and iRBCs result in sub-optimal yields. Inexpensive high-strength neodymium magnets offer a better option, resulting in higher yields with no detrimental effects on parasite viability.