Affinity Purification Method Research Articles

Latrophilin-2 mediates fluid shear stress mechanotransduction at endothelial junctions

Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function, and disease. A complex of PECAM-1, VE-cadherin, VEGF receptors (VEGFRs), and Plexin D1 located at cell–cell junctions mediates many of these events. However, available evidence suggests that another mechanosensor upstream of PECAM-1 initiates signaling. Hypothesizing that GPCR and Gα proteins may serve this role, we performed siRNA screening of Gα subunits and found that Gαi2 and Gαq/11 are required for activation of the junctional complex. We then developed a new activation assay, which showed that these G proteins are activated by flow. We next mapped the Gα residues required for activation and developed an affinity purification method that used this information to identify latrophilin-2 (Lphn2/ADGRL2) as the upstream GPCR. Latrophilin-2 is required for all PECAM-1 downstream events tested. In both mice and zebrafish, latrophilin-2 is required for flow-dependent angiogenesis and artery remodeling. Furthermore, endothelial-specific knockout demonstrates that latrophilin plays a role in flow-dependent artery remodeling. Human genetic data reveal a correlation between the latrophilin-2-encoding Adgrl2 gene and cardiovascular disease. Together, these results define a pathway that connects latrophilin-dependent G protein activation to subsequent endothelial signaling, vascular physiology, and disease.

A glial circadian gene expression atlas reveals cell type and disease-specific reprogramming in response to amyloid pathology or aging.

While circadian rhythm disruption may promote neurodegenerative disease, how aging and neurodegenerative pathology impact circadian gene expression patterns in different brain cell types is unknown. Here, we used translating ribosome affinity purification methods to define the circadian translatomes of astrocytes, microglia, and bulk cerebral cortex, in healthy mouse brain and in the settings of amyloid-beta plaque pathology or aging. Our data reveal that glial circadian translatomes are highly cell type-specific and exhibit profound, context-dependent reprogramming of rhythmic transcripts in response to amyloid pathology or aging. Transcripts involved in glial activation, immunometabolism, and proteostasis, as well as nearly half of all Alzheimer Disease (AD)-associated risk genes, displayed circadian oscillations, many of which were altered by pathology. Amyloid-related differential gene expression was also dependent on time of day. Thus, circadian rhythms in gene expression are cell- and context dependent and provide important insights into glial gene regulation in health, AD, and aging.

Isolation of yeast mitochondria by affinity purification using magnetic beads.

Isolated mitochondria have been widely utilized in various model organisms to investigate the diverse functions of the organelle. Techniques such as differential centrifugation, density gradient ultracentrifugation and antibody-coated magnetic beads are employed for isolation of the organelle from whole cells. However, mitochondria isolated using differential centrifugation are often contaminated with other organelles; isolation using density gradient ultracentrifugation can reduce contamination but is time-intensive and requires large amounts of starting materials; and mitochondria isolated using antibody-coated magnetic beads are irreversibly bound to the beads. Here, we provide a step-by-step protocol for the isolation of highly pure mitochondria from Saccharomyces cerevisiae using a magnetic bead affinity purification method that overcomes these limitations. This protocol describes how to isolate mitochondria, tagged by insertion of 6 histidines (6xHis) into the chromosomal copy of the TOM70 (Translocase of outer membrane 70) gene using Ni-NTA (nickel(II) nitrilotriacetic acid) paramagnetic beads, and the subsequent release of mitochondria from the beads using a buffer containing imidazole. We provide examples of expected results, highlighting the purity, integrity and import activity of isolated mitochondria. These affinity-purified mitochondria are intact and functional, containing less contamination with cytosol and other organelles compared to mitochondria isolated by other methods. Our method is adaptable and can be applied to other model organisms that can be genetically manipulated using CRISPR or other methods.

Transcriptional regulation of the fidaxomicin gene cluster and cellular development in Actinoplanes deccanensis YP-1 by the pleiotropic regulator MtrA

ABSTRACT Fidaxomicin is an 18-membered glycosidic macrolide polyketide that was clinically used in the treatment of Clostridium difficile infection in 2011. Although the biosynthetic mechanism and pathway-specific regulator of fidaxomicin were well elaborated, little is known about the pleiotropic regulators outside the fidaxomicin gene cluster. To further reveal the transcriptional regulatory mechanism of fidaxomicin biosynthesis in Actinoplanes deccanensis YP-1, we used the 5′-biotin-labeled fadR1 promoter ( fadS5R1p ) as a probe to affinity isolate the fadS5R1p -interactive protein MtrA, a response regulator of the two-component system MtrAB, from the proteome of mycelia. MtrA is bound directly to fadS5R1p to positively regulate gene cluster expression and fidaxomicin production. Fidaxomicin production was improved by 37% in the mtrA overexpressed strain. Furthermore, we revealed that MtrA tends to bind GTKAYS motifs and influence genes involved in secondary metabolites, phosphate transport, methionine transport, nitrogen metabolism, drug transport, membrane properties, etc. Overall, for the first time at the transcriptional level, we comprehensively revealed a pleiotropic regulator, MtrA, that not only directly mediates the FadR1-specific pathway to regulate the proper production of fidaxomicin but also participates in bacterial growth and development. IMPORTANCE Cascade regulation networks are almost present in various kinds of microorganisms, but locating and systematically elucidating specific pleiotropic regulators related to a certain gene cluster can be a tricky problem. Here, based on the promoter of the fidaxomicin pathway-specific regulator FadR1, we utilized a “DNA to Proteins” affinity purification method and captured a global regulator MtrA, which positively regulates fidaxomicin biosynthesis. In the mtrA overexpressed strain, the production of fidaxomicin was improved by 37% compared to the native strain. Then, we combined the “Protein to DNAs” affinity purification method (DAP-seq) with the results of RNA-seq and systematically elucidated the primary and secondary metabolic processes in which MtrA directly or indirectly participates. Thus, our work brought up a new way to improve fidaxomicin production from the perspective of global regulation and analyzed the regulatory mechanism of MtrA. Meanwhile, we provided a novel methodology for the research of cascade regulation networks and vital secondary metabolites.

New tools can propel research in lysosomal storage diseases

Historically, the clinical manifestations of lysosomal storage diseases offered an early glimpse into the essential digestive functions of the lysosome. However, it was only recently that the more subtle role of this organelle in the dynamic regulation of multiple cellular processes was appreciated. With the need for precise interrogation of lysosomal interplay in health and disease comes the demand for more sophisticated functional tools. This demand has recently been met with 1) induced pluripotent stem cell-derived models that recapitulate the disease phenotype in vitro, 2) methods for lysosome affinity purification coupled with downstream omics analysis that provide a high-resolution snapshot of lysosomal alterations, and 3) gene editing and CRISPR/Cas9-based functional genomic strategies that enable screening for genetic modifiers of the disease phenotype. These emerging methods have garnered much interest in the field of neurodegeneration, and their use in the field of metabolic disorders is now also steadily gaining momentum. Looking forward, these robust tools should accelerate basic science efforts to understand lysosomal dysfunction distal to substrate accumulation and provide translational opportunities to identify disease-modifying therapies.

PB1109 Development and Evaluation of an Aptamer-Based Affinity Purification Method for Activated Protein C (APC)

PB1109 Development and Evaluation of an Aptamer-Based Affinity Purification Method for Activated Protein C (APC)

Preparation of HSV-IgM human-mouse chimeric antibody and development of stable recombinant cell line

In order to achieve large-scale production of HSV-IgM (HSV1, HSV2) human-mouse chimeric antibody in vitro, the gene sequence of the corresponding hybridoma cell was harvested by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) technique to clone the chimeric antibody into eukaryotic expression vectors, and express the target proteins in CHO-S cells. At the same time, the screening process of stable cell lines was optimized, and the pressure conditions of pool construction stage and monoclonal screening stage were explored. Finally, the target protein was purified by protein L affinity purification method and the biological activity was detected. The recombinant IgM antibodies, HSV1 and HSV2, weighted at 899 kDa and 909 kDa respectively, were prepared. The optimal screening pressure was 20P200M (the first phase of pressure) and 50P1000M (the second phase of pressure). The final titer for the monoclonal expression of HSV1-IgM and HSV2-IgM was 1 620 mg/L and 623 mg/L, respectively. This study may facilitate the development of quality control products of HSV1 and HSV2 IgM series recombinant antibodies as well as efficient expression of IgM subtype antibodies in vitro.

Development of a liquid chromatography-based versatile bioanalysis for bevacizumab based on pretreatment combining aptamer affinity purification and centrifugal ultrafiltration concentration.

We report on the development of a versatile and accurate bioanalytical method for bevacizumab using a pretreatment method combining affinity purification with anti-idiotypic DNA aptamers and centrifugal ultrafiltration concentration, followed by liquid chromatography (LC)-fluorescence analysis. An affinity purification method using Sepharose beads as an affinity support removed immunoglobulin G and a large amount of coexisting substances in the serum sample. Purified bevacizumab was separated as a single peak by conventional LC and detected fluorometrically, showing good linearity (R2 = 0.999) in the range of 5-200μg/mL, sufficient to analyze bevacizumab concentrations in the blood of bevacizumab-treated patients. By combining this purification method with a concentration method using a centrifugal filtration device that inhibits non-specific adsorption of bevacizumab, the quantitative range was reduced by a factor of 10 while showing good linearity (R2 = 0.999) in the 0.5-20μg/mL range. The developed analytical method is expected to be used not only for general bioanalysis of therapeutic mAbs in clinical settings, but also for next-generation antibody drugs that show drug efficacy at low concentrations and for analysis of trace samples.

End-to-End Semi-automated Mid-scale Protein Screening Platform for Drug Discovery Research.

The drug discovery landscape is ever-evolving and constantly demands revolutionary technology advancements in protein expression and production laboratories. We have built a higher-throughput mid-scale semi-automated protein expression and screening platform to accelerate drug discovery research. The workflow described here enables comprehensive expression and purification screening assessment of challenging or difficult-to-express recombinant proteins in a fast and efficient manner by delivering small but sufficient amounts of high-quality proteins. The platform has been implemented for a wide range of applications that include identification of optimal constructs and chaperones for poorly expressing proteins, assessment of co-expression partners for expressing stable multiprotein complexes, and suitable buffer/additive screening for insoluble or aggregation-prone proteins. The approach allows parallel expression, purification, and characterization of 24 different samples using co-infection or a polycistronic approach in insect cells and enables parallel testing of multiple parameters to improve protein yields. The strategy has been successfully adopted for screening intracellular and secreted proteins in Escherichia coli, mammalian transient expression, and baculovirus expression vector systems. Proteins purified from this platform are used for several structural and functional screens, such as negative staining, biochemical activity assays, mass spectrometry, surface plasmon resonance, and DNA-encoded chemical library screens. In this article, for simplicity, we have focused on detailed expression and purification screening of intracellular protein complexes from insect cells. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Baculovirus generation via homologous recombination Support Protocol 1: Anti-glycoprotein 64 antibody assay Basic Protocol 2: Generation of insect cell biomass expressing target protein(s) Basic Protocol 3: Mid-scale affinity purification Support Protocol 2: Automated method for affinity purification on Hamilton STAR Basic Protocol 4: Size exclusion chromatography Support Protocol 3: Chromeleon 7 operation on Vanquish Duo.

Chitin‐ and Streptavidin‐Mediated Affinity Purification Systems: A Screening Platform for Enzyme Discovery

AbstractAffinity purification of recombinant proteins is an essential technique in biotechnology. However, current affinity purification methods are very cost‐intensive, and this imposes limits on versatile use of affinity purification for obtaining purified proteins for a variety of applications. To overcome this problem, we developed a new affinity purification system which we call CSAP (chitin‐ and streptavidin‐mediated affinity purification) for low‐cost purification of Strep‐tag II fusion proteins. The CSAP system is designed to utilize commercially available chitin powder as a chromatography matrix, thereby significantly improving the cost‐efficiency of protein affinity purification. We investigated the CSAP system for protein screening in 96‐well format as a demonstration. Through the screening of 96 types of purified hemoproteins, several proteins capable of the catalytic diastereodivergent synthesis of cyclopropanes were identified as candidates for an abiotic carbene transfer reaction.

Chitin- and Streptavidin-Mediated Affinity Purification Systems: A Screening Platform for Enzyme Discovery.

Affinity purification of recombinant proteins is an essential technique in biotechnology. However, current affinity purification methods are very cost intensive, and this imposes limits on versatile use of affinity purification for obtaining purified proteins for a variety of applications. To overcome this problem, we developed a new affinity purification system which we call CSAP (chitin- and streptavidin-mediated affinity purification) for low-cost purification of Strep-tag II fusion proteins. The CSAP system is designed to utilize commercially available chitin powder as a chromatography matrix, thereby significantly improving the cost-efficiency of protein affinity purification. We investigated the CSAP system for protein screening in 96-well format as a demonstration. Through the screening of 96 types of purified hemoproteins, several proteins capable of the catalytic diastereo-divergent synthesis of cyclopropanes were identified as candidates for an abiotic carbene transfer reaction.

MP14-07 DEVELOPMENT OF A LIQUID BIOPSY USING EXTRACELLULAR VESICLES TO ASSESS THE SYSTEMIC T CELL IMMUNE LANDSCAPE IN BLADDER CANCER

You have accessJournal of UrologyCME1 Apr 2023MP14-07 DEVELOPMENT OF A LIQUID BIOPSY USING EXTRACELLULAR VESICLES TO ASSESS THE SYSTEMIC T CELL IMMUNE LANDSCAPE IN BLADDER CANCER Karen Doersch, Samuel Walker, Thomas Osinski, Jonathan Flax, and James McGrath Karen DoerschKaren Doersch More articles by this author , Samuel WalkerSamuel Walker More articles by this author , Thomas OsinskiThomas Osinski More articles by this author , Jonathan FlaxJonathan Flax More articles by this author , and James McGrathJames McGrath More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Urothelial carcinoma (UC) is commonly treated with therapeutics that target the immune system, such as checkpoint immunotherapy or Bacillus Calmette-Guerin (BCG). While assessment of T cell function is of utility in predicting response to these therapies, current methods for evaluating systemic T cell immunity or intratumoral immune behavior are limited. As antigenically activated T cells produce extracellular vesicles (EVs) carrying markers of cell subtype and activation state and these are found in the blood, we hypothesized that serum T cell derived EVs could be used to interrogate the systemic antitumor T cell response. The purpose of this study was to evaluate a novel microfluidic strategy ‘catch and display for liquid biopsy’ [CAD-LB] for the rapid assessment of T cell biomarkers on individual EVs from T cell conditioned medium or blood. METHODS: T cell derived EVs from cultured cells. We hypothesized that T cell derived EVs have markers of T cell function that mirror their cell of origin. To test this, we assessed markers of activation and exhaustion on EVs secreted by T cells. Activation and exhaustion were induced in vitro by stimulation with anti-CD3/CD28 beads, mimicking the in vivo stimulation of T cells in an antigen-rich tumor environment. T cell subtype and activation state were assessed by CAD-LB and flow cytometry. T cell markers on serum EVs. We developed an affinity purification method for CD3+ EVs and subsequently evaluated the level of T cell biomarkers on these using CAD-LB in UC specimens from patients. RESULTS: 1. T cell marker expression on EVs from conditioned medium from resting, activated and exhausted cells followed the pattern present on the EV producing T cells. 2. T cell derived EVs from UC patient serum identified markers of activated and functionally exhausted cells, indicating that we can capture the full range of T cell functionality with CAD-LB. 3. Detected T cell EVs with markers of non-circulating tissue-resident cells (present in tumor, secondary lymphoid and peripheral tissue) demonstrate that EVs capture the systemic immune responses. CONCLUSIONS: T cell derived EVs capture the phenotype and identity of their cell of origin. CAD-LB detects a range of T cell populations in serum. Future work will test if T cell EVs capture tumor and systemic antitumor immunity, which may help identify UC patients more likely to respond to immunotherapy. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e184 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Karen Doersch More articles by this author Samuel Walker More articles by this author Thomas Osinski More articles by this author Jonathan Flax More articles by this author James McGrath More articles by this author Expand All Advertisement PDF downloadLoading ...

Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome.

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method was performed for the subsequent paired interrogation of the cell-specific ovarian transcriptome and epigenome using a novel transgenic NuTRAP mouse model. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes, the NuTRAP expression system was targeted to stromal cells using a Cyp17a1-Cre driver. The induction of the NuTRAP construct was specific to ovarian stromal fibroblasts, and sufficient DNA and RNA for sequencing studies were obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with an available Cre line.

Cation affinity purification of histidine-tagged proteins.

Protein purification is a basic technology in both biological research and industrial production, and efficient, convenient, economical, and environmentally friendly purification methods have always been pursued. In this study, it was found that alkaline earth metal cations (Mg2+, Ca2+) and alkali metal cations (Li+, Na+, K+) and even nonmetal cations (e.g., NH4+, imidazole, guanidine, arginine, lysine) can precipitate multi-histidine-tagged proteins (at least two tags in a whole protein) at low salts concentrations that are 1-3 orders of magnitude lower than salting-out, and precipitated proteins could be dissolved at moderate concentration of corresponding cation. Based on this finding, a novel cation affinity purification method was developed, which requires only three centrifugal separations to obtain highly purified protein with purification fold similar to that of immobilized metal affinity chromatography. The study also provides a possible explanation for unexpected protein precipitation and reminds researchers to consider the influence of cations on the experimental results. The interaction between histidine-tagged proteins and cations may also have broad application prospects. KEY POINTS: • Histidine-tagged proteins can be precipitated by low-concentrations common cations • A novel nonchromatographic protein purification method was developed • Purified protein can be obtained in pellet form by only three centrifugations.

Interactomes of Glycogen Synthase Kinase-3 Isoforms.

Functional differentiation of the two isoforms of the protein-serine/threonine kinase, glycogen synthase kinase-3 (GSK-3), is an unsettled area of research. The isoforms are highly similar in structure and are largely redundant, though there is also evidence for specific roles. Identification of isoform-specific protein interactors may elucidate the differences in function and provide insight into isoform-selective regulation. We therefore sought to identify novel GSK-3 interaction partners and to examine differences in the interactomes of the two isoforms using both affinity purification and proximity-dependent biotinylation (BioID) mass spectrometry methods. While the interactomes of the two isomers are highly similar in HEK293 cells, BioID in HeLa cells yielded a variety of preys that are preferentially associated with one of the two isoforms. DCP1B, which favored GSK-3α, and MISP, which favored GSK-3β, were evaluated for reciprocal interactions. The differences in interactions between isoforms may help in understanding the distinct functions and regulation of the two isoforms as well as offer avenues for the development of isoform-specific strategies.

Protein-Protein Interaction Network Mapping by Affinity Purification Cross-Linking Mass Spectrometry (AP-XL-MS) based Proteomics.

Protein-protein interactions (PPIs) are the physical interactions formed among proteins. These interactions are primarily functional, i.e., they arise from specific biomolecular events, and each interaction interface serves a specific purpose. A significant number of methods have been developed for protein interactions in the field of proteomics in the last decade. Advanced mass spectrometry technology significantly contributed to the development of these methods. The rapid advancement of groundbreaking MS technology has greatly aided themapping ofprotein interaction from large-data sets comprehensively. This chapter describes the affinity purification (AP) mass spectrometry (MS)-based methods combined with chemical cross-linking (XL) of protein complexes. This chapter includes sample preparation methods involving cell culture, cell treatments with ligands, drugs, and cross-linkers, protein extractions, affinity purification, sodium dodecyl sulfate (SDS) polyacrylamide gel separation, in-solution or in-gel digestion, liquid-chromatography, and mass spectrometry analysis of samples (LC-MS/MS). Application of a cleavable cross-linker, dual cleavable cross-linking technology (DUCCT) in combination with the affinity purification (AP) method has also been described. Methods for data analysis using unmodified and cross-linked peptide analysis arediscussed.

Adsorption of ice-binding proteins onto whole ice crystal surfaces does not necessarily confer a high thermal hysteresis activity

Many psychrophilic microorganisms synthesize ice-binding proteins (IBPs) to survive the cold. The functions of IBPs are evaluated by the effect of the proteins on the nonequilibrium water freezing-point depression, which is called “thermal hysteresis (TH)”, and the inhibitory effect of the proteins on the growth of larger ice crystals, which is called “ice recrystallization inhibition (IRI)”. To obtain mechanical insight into the two activities, we developed a modified method of ice affinity purification and extracted two new IBP isoforms from Psychromyces glacialis, an Arctic glacier fungus. One isoform was found to be an approximately 25 kDa protein (PsgIBP_S), while the other is a 28 kDa larger protein (PsgIBP_L) that forms an intermolecular dimer. Their TH activities were less than 1 °C at millimolar concentrations, implying that both isoforms are moderately active but not hyperactive IBP species. It further appeared that both isoforms exhibit high IRI activity even at submicromolar concentrations. Furthermore, the isoforms can bind to the whole surface of a hemispherical single ice crystal, although such ice-binding was generally observed for hyperactive IBP species. These results suggest that the binding ability of IBPs to whole ice crystal surfaces is deficient for hyperactivity but is crucial for significant IRI activity.

Trap-TRAP, a Versatile Tool for Tissue-Specific Translatomics in Zebrafish.

Developmental and physiological processes depend on the transcriptional and translational activity of heterogeneous cell populations. A main challenge in gene expression studies is dealing with this intrinsic complexity while keeping sequencing efficiency. Translating ribosome affinity purification (TRAP) methods have allowed cell-specific recovery of polyribosome-associated RNAs by genetic tagging of ribosomes in selected cell populations. Here we combined the TRAP approach with adapted enhancer trap methods (trap-TRAP) to systematically generate zebrafish transgenic lines suitable for tissue-specific translatome interrogation. Through the random integration of a GFP-tagged version of the large subunit ribosomal protein L10a (EGFP-Rpl10a), we have generated stable lines driving expression in a variety of tissues, including the retina, skeletal muscle, lateral line primordia, rhombomeres, or jaws. To increase the range of applications, a UAS:TRAP transgenic line compatible with available Gal4 lines was also generated and tested. The resulting collection of lines and applications constitutes a resource for the zebrafish community in developmental genetics, organ physiology and disease modelling.

Human Auto-IgG Purification from High Volume Serum Sample by Protein G Affinity Purification.

Immunoglobulins are proteins produced by the immune system, which bind specifically to the antigen that induced their formation and target it for destruction. Highly purified human immunoglobulins are commonly used in research laboratories for several applications, such as in vitro to obtain hybridomas and in vivo animal immunisation. Several affinity purification methods are used to purify immunoglobulins from human serum, such as protein A/G Sepharose beads, polyethylene glycol, and caprylic acid ammonium sulphate precipitation. Here, we provide a detailed protocol for purification of high-quality IgG from human serum, using affinity chromatography with protein G. The protocol is divided into four main steps (column preparation, serum running, wash, and elution) for IgG purification, and two extra steps (protein dialysis and sucrose concentration) that should be performed when buffer exchange and protein concentration are required. Several IgG affinity purification methods using protein A or G are available in the literature, but protein A has a higher affinity for rabbit, pig, dog, and cat IgG, while protein G has a higher affinity for mouse and human IgG. This affinity-based purification protocol uses protein G for a highly specific purification of human IgG for animal immunization, and it is particularly useful to purify large amounts of human IgG. This protocol was validated in: Pain (2019), DOI: 10.1097/j.pain.0000000000001662 Graphical abstract IgG purification protocol. The IgG purification protocol consists of four main steps (column preparation, serum running, wash, and elution) and two extra steps (protein dialysis and concentration). a. Diluted serum is added to the protein G beads and IgG binds to the Fc receptors on protein G beads. b. Beads are washed in Hartman's solution to fully remove the complex protein mixture (multicolour shapes, as depicted in the graphical abstract). c. IgG (orange triangles, as depicted in the graphical abstract) are removed from protein G with glycine and collected in Tris buffer. d. The IgG is transferred into a semi-permeable membrane ('snake skin') and allowed to dialyse overnight for buffer exchange with a physiological solution (Hartmann's).

A Novel Method of Magnetic Nanoparticles Functionalized with Anti-Folate Receptor Antibody and Methotrexate for Antibody Mediated Targeted Drug Delivery.

Therapeutic effects of anticancer medicines can be improved by targeting the specific receptors on cancer cells. Folate receptor (FR) targeting with antibody (Ab) is an effective tool to deliver anticancer drugs to the cancer cell. In this research project, a novel formulation of targeting drug delivery was designed, and its anticancer effects were analyzed. Folic acid-conjugated magnetic nanoparticles (MNPs) were used for the purification of folate receptors through a novel magnetic affinity purification method. Antibodies against the folate receptors and methotrexate (MTX) were developed and characterized with enzyme-linked immunosorbent assay and Western blot. Targeting nanomedicines (MNP-MTX-FR Ab) were synthesized by engineering the MNP with methotrexate and anti-folate receptor antibody (anti-FR Ab). The cytotoxicity of nanomedicines on HeLa cells was analyzed by calculating the % age cell viability. A fluorescent study was performed with HeLa cells and tumor tissue sections to analyze the binding efficacy and intracellular tracking of synthesized nanomedicines. MNP-MTX-FR Ab demonstrated good cytotoxicity along all the nanocomposites, which confirms that the antibody-coated medicine possesses the potential affinity to destroy cancer cells in the targeted drug delivery process. Immunohistochemical approaches and fluorescent study further confirmed their uptake by FRs on the tumor cells’ surface in antibody-mediated endocytosis. The current approach is a useful addition to targeted drug delivery for better management of cancer therapy along with immunotherapy in the future.