Iminodiacetic acid is a dicarboxylic amino acid with the chemical formula C4H7NO2.
It is used as a chelating agent in various industrial and research applications, including water treatment, metal extraction, and as a precurser for the synthesis of other organic compounds.
Iminodiacetic acid has a wide range of uses in biochemical research, where it can be employed to purify and immobilize proteins and other biomolecules.
Researchers can optimize their iminodiacetic acid experiments with PubCompare.ai, an AI-driven comparison tool that helps identify the most reproducible and accruate protocols from scientific literature, preprints, and patents.
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Most cited protocols related to «Iminodiacetic acid»
The preparation of planar lipid bilayers is detailed elsewhere (Grakoui et al., 1999 (link); Carrasco et al., 2004 (link)). Bilayers were prepared that contained biotin lipids to which biotinylated ICAM-1 and antigens were attached through streptavidin. In brief, PC10-BSA, NIP16-BSA, and the mouse ICAM-1/huFc chimera protein (R&D Systems) were biotinylated with EZ-link sulfo-NHS-LC-biotin (Thermo Fisher Scientific). An aliquot of each was labeled with sulfo-NHS–functionalized fluorophores (Invitrogen) to allow monitoring of the mobility of the lipid-anchored proteins in the lipid bilayers. Biotin-labeled small unilamellar lipid vesicles were prepared by mixing a 100:1 molar ratio of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-cap-biotin (Avanti Polar Lipids, Inc.). The lipid mixture was sonicated and resuspended in PBS at a lipid concentration of 5 mM. Aggregated lipid vesicles were cleared by ultracentrifugation and filtering. Bilayers were formed in Lab-Tek chambers (Thermo Fisher Scientific) in which the coverglasses were replaced with nanostrip-washed coverslips. The coverslips were incubated with 0.1 mM biotin-labeled small unilamellar lipid vesicles in PBS for 10 min. After washing with 20 ml PBS, the bilayer was incubated with 2.5 μg/ml streptavidin for 10 min, and excess streptavidin was removed by washing with 20 ml PBS. The bilayers were incubated for 20 min with 0.5 μg/ml of biotinylated mouse ICAM-1, and excess ICAM-1 was removed by washing. The streptavidin- and ICAM-1–containing planar lipid bilayers were incubated with 0.75 μg/ml of biotinylated PC-BSA or NIP-BSA. The unbound excess of antigen was removed by washing with 20 ml PBS. The mobility of ICAM-1 and antigens in the lipid bilayers was confirmed by analyses of the proteins labeled with fluorescent dyes. Alternatively, NIP- and ICAM-1–containing planar lipid bilayers were prepared by fusing small unilamellar lipid vesicles with a clean glass coverslip surface as described previously (Brian and McConnell, 1984 (link)) using 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyl iminodiacetic acid) succinyl] in nickel salt (Avanti Polar Lipids, Inc.) at a 10:1 ratio. Small unilamellar vesicles were obtained by sonication and clarified by ultracentrifugation and filtering. Glass coverslips were cleaned in Nanostrip (Cyantek), washed, and dried. Lipid bilayers were prepared from a 0.1-mM lipid solution on the coverslips attached to the bottom of Lab-Tek imaging chambers. After excess lipids were washed away, histidine-tagged antigens and ICAM-1 were bound. Before imaging, chambers were washed with HBSS supplemented with 1% FCS. NIP14-BSA was prepared as described previously (Tolar et al., 2005 (link)) and conjugated to a cystein-containing peptide terminated with a 12-histidine tag (ASTGTASACTSGASSTGSH12) using SMCC (Thermo Fisher Scientific) according to the manufacturer's protocols. Recombinant ICAM-1 tagged with a 12-histidine tag was a gift from J. Huppa (Stanford University, Palo Alto, CA). Conjugation of NIP14-BSA to succinimidyl AlexaFluor647 and ICAM-1-H12 to AlexaFluor488 (both obtained from Invitrogen) was performed according to the manufacturer's protocols.
Sohn H.W., Tolar P, & Pierce S.K. (2008). Membrane heterogeneities in the formation of B cell receptor–Lyn kinase microclusters and the immune synapse. The Journal of Cell Biology, 182(2), 367-379.
Diffusive gels with a thickness of 0.8 mm were prepared according to standard methods.7 We tried to develop a Zr-hydroxide and SPR-IDA containing gel based on Ding et al.,26 (link) who describe a Zr-hydroxide-polyacrylamide resin gel for measuring phosphate. However, the addition of Zr-hydroxide to the acrylamide gel solution, both as dried and ground powder as described or as moist slurry, caused rapid acrylamide polymerization and yielded a visibly inhomogeneous Zr-hydroxide gel. Despite our efforts to improve the formulation of the gel solution, we did not succeed in obtaining homogeneous resin distribution using polyacrylamide hydrogels. It is possible that the Zr-hydroxide particles interfered with the polymerization process, as group III–VIII transition metals are known for their potential to catalyze polymerization reactions.27 To overcome these problems, a urethane-based hydrogel (Hydromed D4, Advan Source biomaterials, Massachusetts, US), which has not been used for DGT gels so far, served as gel matrix for embedding the Zr-hydroxide and SPR-IDA resins. This gel material is an ether-based hydrophilic urethane polymer which does not require UV curing or a polymerization reaction. The gel is formed upon solvent evaporation. We refer to this novel resin gel as “high resolution mixed binding gel” (HR-MBG) throughout this paper. A total of 15 g of ZrOCl2·xH2O (Alfa Aesar, 99.9985%) was dissolved in 500 mL of laboratory water type 1. Zirconium hydroxide precipitate was prepared by titrating this solution with a 0.1 mol L–1 NaOH (Alfa Aesar, 99.99% metals basis) solution under vigorous stirring until the pH stabilized at 7.0. The solution containing the precipitated Zr-hydroxide was filtered using a vacuum flask and a Buechner-funnel (VWR 454, quantitative filter paper). Subsequently, the precipitated Zr-hydroxide was washed by adding 5 L of laboratory water type 1 to the funnel and sucking the water off until only the slurry remained on the filter. The moist precipitate was transferred into acid-washed containers and stored at 6 °C. A batch of 15 g of ZrOCl2·xH2O yields approximately 140 g of precipitate (wet weight). Ten grams of the hydrogel material Hydromed D4 was crushed to ∼5 mm pieces and dissolved in 100 mL of an ethanol (Sigma, Aldrich, puriss)-laboratory water type 1 solution (10:1, v/v). Fifteen grams of the Zr-hydroxide slurry was transferred into an acid washed polypropylene container. Approximately 90 mL of Hydromed solution was added to yield 100 mL of the Zr-hydroxide–Hydromed mixture. This mixture was homogenized with a dispersing device (Ultra-Turrax T10 Basic, IKA-Werke GmbH & CO. KG, Staufen, Germany) at ∼20,000 rpm for 5 min. One milliliter of suspended particulate reagent iminodiacetic acid (SPR-IDA; CETAC Technologies, Nebraska, US) resin was added to 9 mL of Zr-hydroxide–Hydromed solution and vigorously shaken by hand for 3 min. This solution was then fixed in an overhead-shaker and rotated at 2–3 rpm overnight to eliminate air bubbles from the viscous gel solution. An acid-washed plastic spacer with a thickness of 0.25 mm was arranged on a glass plate in a U-shape (approximately 6 × 20 cm in size) and fixed on the outer side with small strips of adhesive tape. A layer of hydrogel was coated onto a glass plate by consecutively applying three thin layers of gel on top of each other. Therefore, approximately 3 mL of bubble-free gel solution was poured into the spacer and evenly distributed within using a second glass plate as a coating tool. The glass plate with the freshly coated gel solution was put into an oven at 80 °C until the gel was dry (approximately 10 min). Afterward, the hot glass plate was allowed to cool to room temperature in a clean bench, and the coating process was repeated two more times to achieve a triple coating. The triple-coated gel was allowed to cool to room temperature. The spacer was removed, and the outer 2 mm of the gel sheet was cut off using a razor blade to remove areas with inhomogeneous resin distribution along the gel edges. The gel sheet with the glass plate was put into 5 L laboratory water type 1 for at least 4 h to hydrate. Afterward, it was gently detached from the glass plate using tweezers and placed in a fresh water bath of 5 L laboratory water type 1 for full hydration for 24 h. With this procedure, a 100 μm-thick gel with highly homogeneous resin distribution was produced. The thin gel is a result of solvent removal during the drying process where the solvents, especially ethanol, are evaporated at 80 °C. A circular metal die-cutter had to be used to cut gel discs, as the gel is very stable and tear-proof. Teflon-coated razor blades were used for cutting rectangular gel pieces. The hydrated gel was stored in 10 mmol L–1 NaNO3 solution at 6 °C. DGT devices as provided by DGT Research Ltd. (Lancaster, UK) were used for the solution experiments. These samplers are designed to host a sampling setup consisting of 0.4 mm of resin gel, 0.8 mm of diffusive gel, and 0.14 mm of protective membrane. To compensate for the HR-MBG gels being thinner than common resin gels, a 0.4 mm plastic spacer was placed at the bottom of the sampler. This assembly was used throughout the solution experiments. If only the gel discs were deployed without the sampling device, this is noted in the experimental description.
Kreuzeder A., Santner J., Prohaska T, & Wenzel W.W. (2013). Gel for Simultaneous Chemical Imaging of Anionic and Cationic Solutes Using Diffusive Gradients in Thin Films. Analytical Chemistry, 85(24), 12028-12036.
Expression, purification, and reconstitution of Fluc channels were as described in detail27 (link),28 (link). In the final purification step, Fluc protein was collected from a S200 size-exclusion column equilibrated in 100 mM NaF (or NaCl for zero-F- preps), 10 mM Hepes pH 7.0, 5 mM n-decylmaltoside (DM). Bpe constructs carried two functionally neutral mutations to enhance expression, R29K/E94S or, for Hg labeling, R29K/E94C. Hg labeling was achieved by incubating Bpe with a 3-fold molar excess of Hg(II) acetate for 30 minutes between the Co-affinity and size exclusion columns. Ec2 constructs bore a single functionally neutral, expression-enhancing mutation, R25K, and, for selenomethionine incorporation, an additional methionine was introduced (A51M) to enhance phasing power. The C-terminal His6 tag was removed from Bpe by treatment with lysine endoproteinase C (Roche)27 (link), but was left on Ec2. Fluc protein was typically reconstituted into liposomes at low density (0.1-0.2 µg protein/mg lipid). For single-channel recording, liposomes were fused into planar lipid bilayers in symmetrical solutions of 300 mM NaF, 15 mM MOPS, pH 7.0, and channels were recorded at 200 mV holding voltage27 (link),29 . Monobodies were expressed in E. coli and purified as described28 (link). N-terminal His6 tags were removed while bound to Talon beads by 16-hr treatment with TEV protease also carrying a His tag; monobodies with cleaved His tags were eluted from the affinity column with 150 mM NaCl, 40 mM Tris-HCl pH 7.5. For the final purification step, the preparation was passed over a S75 size exclusion column in 100 mM NaF (or NaCl), 10 mM HEPES pH 7. Monobodies were used immediately for crystallization or stored in frozen aliquots for channel-blocking experiments. For crystallization from detergent micelles, Fluc protein in solution containing 5 mM DM was concentrated to 10 mg/mL, a maneuver that concentrates the detergent 5-10-fold. Monobody solution (10 mg/mL) was supplemented with 4 mM DM immediately before mixing with channel in a 1.2:1 molar ratio. This protein solution was then mixed with an equal volume of crystallization solutions (0.5 µL for sitting drops in 96-well plates or 1 µL for hanging drops in 24-well plates). Bpe-S7 crystals grew in 3-5 days in crystallization solutions of 36-41% (w/v) polyethylene glycol 550 MME, 0.2 M MgCl2 or CaCl2, 0.1 M Tris, pH 8.5-8.9. Ec2-S9 crystals grew in 10-14 days in crystallization solutions containing 28-32% (w/v) polyethylene glycol 550 MME, 0.05 M LiNO3, 0.1 M N-(2-Acetamido)iminodiacetic acid, pH 6.0-6.7. Crystals were frozen in liquid nitrogen for data collection. For lipidic cubic phase crystallization, Fluc protein concentrated to 10 mg/mL as above was dialyzed overnight to reduce the DM concentration to 10 mM. This was then mixed with monobody solution (10 mg/mL, with 4 mM DM) in a 1:1.2 molar ratio. The protein-laden mesophase was prepared by homogenizing 9.9 monoacylglycerol (monoolein) lipid with protein solution (10 mg/ml) at a weight ratio of 1:1.5 (protein:lipid) using a coupled syringe mixing device at 20°C30 (link). Crystallization trials were carried out at 19°C in 96-well glass sandwich plates with 50 nl mesophase and 0.8 µl precipitant solution using an in meso robot. Crystallization solutions consisted of 22–26% (v/v) polyethylene glycol 500 DME, 0.1 M Na-citrate pH 5.5 +/- 10 mM NaF. Surfboard shaped crystals grew to a maximum size of 100 × 50 × 5 µm in 5-10 days. Wells were opened using a tungsten–carbide glasscutter, and the crystals were harvested using 50-100 µm micromounts (MiTeGen). Crystals were snap-cooled directly in liquid nitrogen prior to data collection on the Diamond Light Source beamlines I24 or I04.
Stockbridge R.B., Kolmakova-Partensky L., Shane T., Koide A., Koide S., Miller C, & Newstead S. (2015). Crystal structures of a double-barrelled fluoride ion channel. Nature, 525(7570), 548-551.
Cloning and Expression of Ferritin Polypeptides—cDNAs containing the sequence of human WT-FTL and human mutant FTL498–499InsTC were introduced into the pET-28a(+) expression vector (Novagen, EMD Chemicals Inc.). The cDNAs were cloned between the BamHI and XhoI sites, downstream from and in-frame with the sequence encoding an N-terminal His6 tag. To eliminate the His6 tag (included in the expression vector), the sequence of the vector was modified by introducing the recognition sequence for cleavage by factor Xa before the coding sequence of the ferritin genes. PCR amplification of the ferritin cDNAs was performed using the upstream primer F1 5′-TGG ATC CAT CGA AGG TCG TAT GAG CTC CCA GAT T-3′ and the downstream primer R1 5′-TTA TGC CTC GAG CCC TAT TAC TTT GCA AGG-3′. F1 contains the factor Xa sequence (underlined). pET-28a(+) carrying WT-FTL and MT-FTL cDNAs was transformed into BL21 (DE3) Escherichia coli (Invitrogen). Transformed cells were grown in Luria broth medium (LB) containing 30 μg/ml kanamycin (Invitrogen) at 37 °C up to an absorbance of 0.9–1.0 at 600 nm. Bacteria were induced to overexpress recombinant proteins by adding 1 mm isopropyl thio-β-d-galactopyranoside (ICN Biotechnologies) for 12 h at 25 °C. Purification of Recombinant WT- and MT-FTL Homopolymers—Cells were harvested by centrifugation and frozen at -80 °C. The cell pellets were suspended in 50 mm sodium phosphate, 500 mm NaCl (pH 7.4), 1 mg/ml lysozyme, and a protease inhibitor mixture (Complete, Roche Applied Science) for 30 min. Bacteria were disrupted by sonication, and the insoluble material was removed by centrifugation at 21,000 × g for 30 min. The soluble fraction was purified by nickel iminodiacetic acid affinity chromatography using an AKTA purifier system (GE Healthcare). Purified protein was eluted with 250 mm imidazole in 50 mm sodium phosphate (pH 7.4), 0.5 m NaCl. Recombinant proteins were diluted with 50 mm Tris and 10% glycerol (v/v) down to an absorbance of 0.5 at 280 nm, and ferritins were cleaved from the His tag by digestion with factor Xa protease (GE Healthcare) (5 units/mg of protein). After being dialyzed against 50 mm Tris, pH 8.0, for 18 h, proteins were further purified by anion exchange chromatography (Mono Q) using a linear NaCl elution gradient in 50 mm Tris (pH 8). Peak fractions were ∼95% pure based on SDS-12% PAGE (Pierce) and Coomassie Blue staining. The efficiency of tag removal was confirmed by N-terminal protein sequencing analysis, and the molecular weight of the recombinant proteins was determined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Protein concentration was determined using the BCA reagent (Pierce) with bovine serum albumin as standard. Gel Filtration Chromatography—Size exclusion chromatography was performed on a Superose 6 10/300 GL column (GE Healthcare) equilibrated with 50 mm Tris, 150 mm NaCl (pH 7.4) using an AKTA purifier. The column was calibrated with gel filtration standards (GE Healthcare). Fractions were detected photometrically, and peak areas and kav values were evaluated using the UNICORN 5.1 software (GE Healthcare). All gel filtration experiments were run at room temperature. Transmission Electron Microscopy (TEM)—Ferritins were fixed using the “single droplet” parafilm protocol. The specimens were dropped onto a 400-mesh carbon/Formvar-coated grid (Nanoprobes) and allowed to absorb to the Formvar for a minimum of 1 min. Excess fluid was removed using filter paper, and the unbound protein was washed, and the grids were placed on a 50-μl drop of Nanovan (Nanoprobes) with the section side downwards. Finally, the grids were dried, placed in the grid chamber, and stored in desiccators before the samples were observed with a Tecnai G2 12 Bio Twin (FEI) transmission electron microscope. Preparation of Apoferritins—Recombinant FTL homopolymers were treated for iron removal as described previously (14 (link)). Briefly, recombinant ferritins were incubated with 1% thioglycolic acid (pH 5.5) and 2,2′-bipyridine, followed by dialysis against 0.1 m phosphate buffer (pH 7.4). We consistently achieve less than five atoms of iron per ferritin 24-mer, as determined by the colorimetric ferrozine-based assay for the quantitation of iron (15 (link)). Iron Loading of Apoferritins—Freshly prepared ferrous ammonium sulfate (0.5–4.5 mm) in 10 mm HCl was added to MT- and WT-FTL apoferritin homopolymers (1 μm) in 0.1 m Hepes buffer (pH 7.4) at room temperature (16 (link)). After 2 h, the samples were centrifuged at 14,000 × g for 15 min. Iron incorporation was initially monitored by measuring absorbance of the supernatants at 310 nm (14 (link), 17 (link)). Iron incorporation into ferritin was more precisely determined by densitometric analysis of Prussian blue staining of supernatants run on nondenaturing gel electrophoresis. Pellets were analyzed by SDS-12% PAGE. Apoferritins were also incubated in a molar ratio 1:3500 with ferrous ammonium sulfate and centrifuged at 14,000 × g for 15 min. Pellets were resuspended in a solution containing 6 mm deferroxamine (DFX), 0.1 m Hepes (pH 7.4) and incubated for 2 h at 24 °C. After centrifugation, supernatants were analyzed by nondenaturing gel electrophoresis. Circular Dichroism Spectroscopy—CD spectra of recombinant apoferritin homopolymers were obtained in 50 mm phosphate buffer (pH 7.4) at 25 °C in a Jasco 810 spectropolarimeter (Jasco Corp.), using a protein concentration of 0.12 and 1.5 μm for far-UV and near-UV, respectively. Far-UV CD spectra were recorded in a 1.0-mm path length cell from 250 to 190 nm with a step size of 0.1 nm and a bandwidth of 1.0 nm. Each spectrum represents the mean of 15 scans. CD spectra of the buffer/cuvette were recorded and subtracted from the protein spectra before averaging. Secondary structure analyses were performed using DICHROWEB (18 (link), 19 ), which allows secondary structure analyses via the software package CDPro (20 (link)). SELCON3 (21 (link)), CONTINLL (22 (link)), and CDSSTR (23 (link)) programs were used for comparing variations in the amount of secondary structure between MT- and WT-FTL homopolymers. Normalized root mean square deviation values of < 0.1 for the three methods meant that the experimental and simulated spectra were in close agreement. Near-UV CD spectra were recorded in a 1.0-cm path length cell from 400 to 250 nm with a step size of 1.0 nm and a bandwidth of 1.5 nm. For all spectra, an average of five scans was obtained. Intrinsic Protein Fluorescence and Thermal Stability Studies of Homopolymers—Fluorescence spectra were recorded using a spectrofluorimeter (PerkinElmer Life Sciences) equipped with a Selecta Ultraterm water bath for temperature control. Apoferritin spectra were obtained with excitation at 280 and 295 nm with 1.5 μm protein in 1-cm path length cells and with 0.1 m phosphate (pH 7.4). Blanks without protein were subtracted from the spectra. Thermal denaturation was induced by increasing the temperature from 20 to 100 °C at a rate of 1 °C/min. To overcome the inherent difficulty in denaturing ferritin, these experiments were performed in 0.1 m phosphate buffer (pH 7.4) containing 4.0 m guanidine hydrochloride (GdnHCl). Homopolymer stability was monitored using the ratio of intrinsic fluorescence emission of 355 over 330 nm with excitation at 295 nm (24 (link), 25 (link)) with a maximum at 330 nm signifying native ferritin (mt and WT) and 355 nm, denatured ferritin. ANS Fluorescence and Binding Studies—Extrinsic fluorescence spectra were recorded using a spectrofluorimeter (PerkinElmer Life Sciences) in 1.0-cm cuvettes at 25 °C. ANS binding to apoferritin homopolymers was monitored through fluorescence enhancement with ANS excitation at 360 nm and emission recorded from 600 to 400 nm. MT-FTL apoferritins were prepared by diluting stock solutions to 1.5 μm in 0.05 m phosphate buffer (pH 7.4). Stock solutions of ANS (Invitrogen) were prepared in water, and the concentration was determined optically at 350 nm using an extinction coefficient of 4950 m-1 cm-1. ANS was added to the diluted ferritin samples and equilibrated for 30 min prior to the measurements, and spectra were background corrected. Binding of ANS to ferritin was quantitated by Scatchard analysis (26 ). Thermolysin Treatment of WT- and MT-FTL Apoferritin Homopolymers—Proteolysis of recombinant MT- and WT-FTL homopolymers was initiated by adding to 10 μg of ferritin a 10-fold concentrated stock solution (36.5 units/mg) of thermolysin (Fluka) in Hepes (0.1 m) (pH 7.0), 10 mm CaCl2 to a final concentration of 0.2 mg/ml. The reaction was stopped by the addition of EDTA (50 mm) and Laemmli sample buffer. Samples treated with thermolysin and controls without thermolysin were boiled and loaded onto SDS-polyacrylamide gels (4–20%) (Pierce). Gels were stained with Coomassie Blue (Total protein) or blotted against the C-terminal antibodies (MT-1283 or WT-1278) (9 (link)) or against the N-terminal antibody D18 (Santa Cruz Biotechnology, Inc), which recognized both polypeptides. Astrocyte Cell Cultures and Iron/Chelator Treatment—Primary cortical astrocyte cultures were prepared from 1-day-old mouse pups according to the procedures of Saneto and De Vellis (27 ) and Cassina et al. (28 (link)), with minor modifications. Pups were obtained from transgenic dams homozygous for the FTL498–499InsTC mutation in C57BL/6J genetic background (29 (link)). Briefly, cerebral cortices were removed, and the tissue was minced and dissociated in 0.25% trypsin (Invitrogen) for 15 min at 37 °C. Cells were collected by centrifugation and plated at a density of 2.0 × 106 cells in 25-cm2 flasks (Corning Glass) in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, Hepes (25 mm), penicillin (100 IU/ml), and streptomycin (100 μg/ml) (Invitrogen). When confluent, cultures were shaken for 48 h at 250 rpm at 37 °C, incubated for another 48 h with 10 μm cytosine arabinoside, and then amplified to 2.5 × 104 cells/cm2 in 75-cm2 flasks (Corning Glass). The astrocyte monolayers were >98% pure as determined by GFAP immunoreactivity. Confluent astrocyte monolayers were changed to Dulbecco's modified Eagle's media devoid of serum prior to treatment. Stock solutions (20 mm) of ferric ammonium citrate (FAC) (Sigma), and 1,10-phenanthroline (Phen) (Sigma) were prepared in distilled water and directly applied to the monolayer at the indicated final concentrations. Each flask was treated with either of the following: (a) vehicle (water) as control group; (b) Phen at 100 μm during 48 h followed by 24 h at 50 μm;(c) FAC 50 μm during 4 days; (d) FAC treatment as in c followed by Phen treatment as in b in the absence of iron. Characterization of Detergent-insoluble MT-FTL Ferritin from Astrocyte Cultures—Cerebral cortical astrocytes cultures were homogenized in lysis buffer (3 ml of 50 mm Tris-HCl (pH 7.4), 1% SDS, 30 units/ml benzonase, 2 mm MgCl2) containing Complete protease inhibitor mixture (Roche Applied Science) and incubated for 15 min at room temperature. Lysates containing equal amounts of protein were ultracentrifuged at 46,000 rpm (TLA 110, Beckman) for 25 min at 4 °C. The supernatant (SDS-soluble) was removed, and the SDS-insoluble pellet was resuspended in lysis buffer and then subjected to another step of centrifugation in the same conditions. The final pellet was resuspended in 5× Laemmli sample buffer and heated for 10 min at 95 °C. The SDS-soluble, -insoluble, and total cell lysates (before SDS extraction) were resolved on 4–20% gradient SDS-PAGE (Pierce) and transferred to nitrocellulose membranes (Amersham Biosciences). Membranes were blocked for1 h in 70 mm Tris-buffered saline, 0.1% Tween 20, and 5% nonfat dry milk, followed by an overnight incubation with polyclonal antibodies (1283) against the MT-FTL polypeptide, as described previously (9 (link), 29 (link)) at 1:10,000. After washing, membranes were incubated with peroxidase-conjugated secondary antibody (GE Healthcare) for 1 h, washed, and developed using the ECL chemiluminescent detection system (GE Healthcare). MT-FTL recombinant polypeptides were loaded and used as positive control.
Sequence comparison between WT- and MT-FTL polypeptide. The wild type FTL polypeptide (WT-FTL) consists of 175 amino acids. The p.Phe167SerfsX26 mutant polypeptide (MT-FTL) has 191 amino acids and a different C-terminal sequence (underlined). The boxes indicate the five α-helical domains in the WT-FTL polypeptide according to Protein Data Bank accession number 2FG4. The mutant C-terminal sequence contains both metal-binding and hydrophobic groups.
MT-FTL polypeptides assemble into 24-mer homopolymers.A, elution profiles of purified WT- and MT-FTL apoferritin homopolymers from a Superose 6 column at pH 7.4 in 0.05 m Tris, 0.15 m NaCl. Retention times for both proteins are shown. Arrows indicate the elution time for the molecular weight markers. B, ultrastructural characterization of WT- and MT-FTL homopolymers by TEM. The dark cores most likely represent Nanovan that has penetrated in some cases the interior of the 24-mers. Bars, 10 nm. C, native PAGE (3–8% (pH7.4)) of 0.5 μm WT- and MT-FTL proteins loaded before the removal of iron and stained with Coomassie Blue (protein staining) and with Prussian blue (iron staining).
Immunofluorescence of Cultured Cells—Astrocyte cultures in Lab-Tek chambered coverglass slides (Nunc) were fixed for 15 min with 4% paraformaldehyde in PBS at 4 °C. Briefly, the slides were washed successively with PBS, permeabilized with 0.1% Triton X-100 for 15 min, and incubated for 1 h at room temperature in blocking solution (0.1% Triton X-100, 2% bovine serum albumin in PBS). The cultures were incubated overnight at 4 °C with the primary antibodies diluted in blocking solution, washed with PBS, and further incubated for 1 h at room temperature with the secondary antibodies diluted in blocking solution. The slides were then washed with PBS, rinsed with distilled water, and mounted with the Prolong Gold antifade mounting reagent (Molecular Probes). Primary antibodies used were monoclonal antibody to GFAP (1:400; Sigma) and polyclonal antibody against MT-FTL (1283). Secondary antibodies used were Alexa 488 Fluor-conjugated goat anti-rabbit and Alexa Fluor 594-conjugated goat anti-mouse (4 μg/ml; Molecular Probes). Images were captured with a Zeiss LSM-510 confocal scanner attached to a Zeiss Axiovert 100 M inverted microscope.
Baraibar M.A., Barbeito A.G., Muhoberac B.B, & Vidal R. (2008). Iron-mediated Aggregation and a Localized Structural Change Characterize Ferritin from a Mutant Light Chain Polypeptide That Causes Neurodegeneration. The Journal of Biological Chemistry, 283(46), 31679-31689.
Planar fluid lipid bilayers were prepared as previously described (Brian and McConnell, 1984 (link); Grakoui et al., 1999 (link); Carrasco et al., 2004 (link)). In brief, Ni-NTA–containing lipid bilayers were prepared using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[N(5-amino-1-carboxypentyl) iminodiacetic acid]-succinyl (nickel salt; DOGS–Ni-NTA; Avanti Polar Lipids, Inc.) in a 9:1 DOPC/DOGS–Ni-NTA ratio. Unilamellar vesicles were formed by sonication of the mixed lipids and clarified by ultracentrifugation and filtering. Glass coverslips were cleaned by Nanostrip (Cyantek), washed, and dried. Lipid bilayers were prepared from 0.1-mM lipid unilamellar vesicle solutions on the coverslip attached to the bottom of Labtek chambers (Thermo Fisher Scientific). NIP or pNP conjugated to the peptide ASTGKTASACTSGASSTGSHis12 (NIP1-His12 or pNP1-His12) and NIP1-His12 and pNP1-His12 coupled to Hylight647 through the cysteine residue were purchased from Anaspec. The hapten–peptide conjugates were HPLC purified and verified by mass spectroscopy with >95% purity. The hapten-coupled peptides were attached to the Ni-NTA–containing lipid bilayer by incubating haptenated peptides (10 or 50 nM) with the bilayer for 20 min at room temperature (RT). Where indicated in the figures, mouse ICAM-1/huFc chimera protein with a 10-nM His12 tag (R&D Systems) was also attached to the lipid bilayers. After washing, the antigen-containing lipid bilayers were used in TIRF imaging. The amount of antigen bound to the bilayer was quantified by titration of the Hylight647-conjugated peptides to resolve single molecules. The concentration of the peptide attached to the bilayer was calculated by a function of where C is the concentration (number of molecules per square micrometer), N is the number of Hylight647-conjugated peptide molecules counted at a single-molecule resolution per square micrometer, and D is the titration rate. In our experimental system, incubating bilayers with a 10-nM haptenated peptide solution resulted in bilayers containing ∼25 molecules/µm2, and incubating with a 50-nM solution resulted in a bilayer containing 100 molecules/µm2 in the planar lipid bilayer.
Liu W., Meckel T., Tolar P., Sohn H.W, & Pierce S.K. (2010). Antigen affinity discrimination is an intrinsic function of the B cell receptor. The Journal of Experimental Medicine, 207(5), 1095-1111.
The twenty-four single recombinant proteins were expressed as previously described (Table S2). All recombinant proteins contain a cluster of six histidine residues at the N- and C-termini. The proteins were purified using one-step metal affinity chromatography with Ni2+ bound to iminodiacetic acid-agarose (Merck, KGaA, Darmstadt, Germany). The purification resulted in electrophoretically homogeneous protein preparations with a purity above 90% (Figure S7). The concentration of purified proteins was determined using Bradford reagent (Merck, KGaA, Darmstadt, Germany) according to the manufacturer’s recommendation.
Ferra B.T., Chyb M., Sołowińska K., Holec-Gąsior L., Skwarecka M., Baranowicz K, & Gatkowska J. (2024). The Development of Toxoplasma gondii Recombinant Trivalent Chimeric Proteins as an Alternative to Toxoplasma Lysate Antigen (TLA) in Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Immunoglobulin G (IgG) in Small Ruminants. International Journal of Molecular Sciences, 25(8), 4384.
Experimental conditions were 2.5 μM protein, 20 mM Tris (pH 7.5), 0.15 M NaCl, 37 °C. GdnHCl denaturation experiments were performed as previously described (7 ) except 1 mM EDTA was added to the apoLECT2 samples and 7.5 μM ZnCl2 and 22.5 μM iminodiacetic acid were added to the holoLECT2 samples to buffer [Zn2+]free at a constant concentration of 6 to 8 μM. Data were fit to the two-state linear extrapolation equation (59 (link)) to obtain thermodynamic parameters. Equilibrium zinc binding and zinc dissociation kinetic experiments were performed as described previously (7 ).
Ha J.H., Xu Y., Sekhon H., Zhao W., Wilkens S., Ren D, & Loh S.N. (2024). Mimicking kidney flow shear efficiently induces aggregation of LECT2, a protein involved in renal amyloidosis. The Journal of Biological Chemistry, 300(5), 107231.
The yeast strain Komagaetella phaffii X-33 transformed with the plasmid previously characterized for the expression of hLF was used, and its production was induced in the YPD selective culture medium (Sigma-Aldrich, St. Louis, MO, USA), incubating at 30 °C for 48 h with stirring at 150 RPM and with aeration. Recombinant proteins were purified from the culture medium using a ProbondTM affinity chromatography system (Thermo Fisher Scientific, Waltham, MA, USA), which uses a polymeric matrix activated with iminodiacetic acid and nickel (nickel is a di- and trivalent metal that allows us to selectively separate recombinant proteins tagged with polyhistidine residues, His Tag). Simple purification of secreted recombinant proteins is possible due to the relatively low levels of native secreted proteins.
Álvarez-Mayorga B.L., Romero-Gómez S., Rosado J.L., Ocampo-Hernández J., Gómez-Guzmán J, & Ortiz-Frade L. (2024). Study of pH and Thermodynamic Parameters via Circular Dichroism Spectroscopy of a Recombinant Human Lactoferrin. Molecules, 29(2), 491.
The PEDV 3CLpro expression vector was obtained by cloning the PEDV nsp5 gene into the Pet-28a (+) vector. This vector was then transformed into the E.coli BL21 (DE3) strain to create recombinant bacteria. The bacteria were cultured at 37°C in Luria Bertani medium until the optical density value at a wavelength of 600 nm (OD600) reaching 0.6–0.8. After that, 0.8 mM isopropyl β-D-thiogalactoside (IPTG) was added to induce protein expression. The bacteria were harvested after incubation at 37°C for 24 h, resuspended in PBS and disrupted. The supernatant was filtered and loaded onto a nickel iminodiacetic acid NUPharose fast flow (Nuptec, Hangzhou, China) to purify the protein. Subsequently, the N and C terminal His-tagged protein was eluted using elution buffer. The protein eluent was desalted by dialysis in PBS buffer and was concentrated by ultrafiltration.
Li Z., Zhu L., Wang L., Huang Y., Zhang Y., Zhao D., Wang L., Yi D., Hou Y, & Wu T. (2024). Identification of two flavonoids antiviral inhibitors targeting 3C-like protease of porcine epidemic diarrhea virus. Frontiers in Microbiology, 15, 1357470.
Protein expression in E. coli and copurification of LSD1Δ124-CoREST1Δ305 (LSD1-COREST) were performed using previously described procedures. 13 (link) LSD1-CoREST crystals were prepared in hanging drop at 20°C in 100 mM N-(2-acetamido)iminodiacetic acid (ADA) pH 6.5, 1.2 M Na/K Tartrate. Crystals were soaked in a solution containing 1 mM bomedemstat for 2 h at 20°C, followed by washing in a reservoir solution supplemented with 20% glycerol for cryo-protection and immediate freezing in liquid nitrogen. X-ray diffraction data were collected on the X06SA beamline at the SLS synchrotron. Data processing and scaling were carried out using XDS and AIMLESS. Structure refinement was performed using REFMAC5. Topologies for the inhibitors were obtained from the PRODRG server. The PDB validation tools were used for structure validation. Final data collection and refinement statistics are shown in Supporting Information S1: Table 1.
Jasmine S., Mandl A., Krueger T.E.G., Dalrymple S.L., Antony L., Dias J., Celatka C.A., Tapper A.E., Kleppe M., Kanayama M., Jing Y., Speranzini V., Wang Y.Z., Luo J., Trock B.J., Denmeade S.R., Carducci M.A., Mattevi A., Rienhoff HY J.r., Isaacs J.T, & Brennen W.N. (2024). Characterization of structural, biochemical, pharmacokinetic, and pharmacodynamic properties of the LSD1 inhibitor bomedemstat in preclinical models. The Prostate, 84(10).
Iminodiacetic acid is a chemical compound commonly used in laboratory equipment. It functions as a chelating agent, capable of forming stable complexes with various metal ions. The core function of iminodiacetic acid is to bind and sequester specific metal ions, facilitating their removal or separation in analytical and purification processes.
1,2-dioleoyl-sn-glycero-3-phosphocholine is a synthetic lipid compound. It is a phospholipid that consists of two oleic acid chains attached to a glycerol backbone, with a phosphocholine headgroup.
DGS-NTA-Ni is a phospholipid that contains a nitrilotriacetic acid (NTA) head group with a nickel (Ni) ion. It is commonly used in protein purification and immobilization applications.
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) is a synthetic phospholipid commonly used in research applications. It is a neutral phospholipid composed of a glycerol backbone, two oleic acid chains, and a choline headgroup. DOPC serves as a building block for model lipid membranes and is widely employed in studies involving membrane structure and function.
DOGS-NTA-Ni2+ is a lipid molecule that can be used in the preparation of liposomes. It contains a nickel-nitrilotriacetic acid (Ni2+-NTA) head group, which can be used to immobilize histidine-tagged proteins on the liposome surface.
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine is a synthetic phospholipid product offered by Avanti Polar Lipids. It is a phosphatidylethanolamine lipid with two oleic acid chains attached to a glycerol backbone and a phosphoethanolamine head group.
The DGS-NTA is a lab equipment product offered by Avanti Polar Lipids. It is a synthetic lipid compound that contains a nitrilotriacetic acid (NTA) headgroup. The core function of the DGS-NTA is to facilitate the immobilization and purification of histidine-tagged proteins.
DOGS-NTA-Ni is a lipid-based reagent used in various biochemical and biophysical applications. It contains the lipid DOGS (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]) and nickel (Ni) ions, which can be used to immobilize His-tagged proteins for affinity-based studies.
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HEPES is a buffering agent commonly used in cell culture and biochemical applications. It helps maintain a stable pH environment for biological processes.
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NaCl is a chemical compound commonly known as sodium chloride. It is a white, crystalline solid that is widely used in various industries, including pharmaceutical and laboratory settings. NaCl's core function is to serve as a basic, inorganic salt that can be used for a variety of applications in the lab environment.
Iminodiacetic acid is a versatile chemical with a wide range of applications. It is commonly used as a chelating agent in various industrial and research settings, such as water treatment, metal extraction, and the synthesis of other organic compounds. In biochemical research, iminodiacetic acid is particularly useful for purifying and immobilizing proteins and other biomolecules.
Iminodiacetic acid exists in different forms, including the free acid and various salt forms, such as sodium iminodiacetate and calcium iminodiacetate. These different variations can have slightly different properties and applications, depending on the specific research or industrial needs.
One of the main challenges with using iminodiacetic acid is ensuring optimal experimental conditions, such as pH, temperature, and concentration. Improper handling or preparation can lead to inconsistent results. Additionally, the chelating properties of iminodiacetic acid can sometimes interfere with other reagents or analytes, requiring careful experimental design.
PubCompare.ai is an AI-driven comparison tool that can greatly assist researchers working with iminodiacetic acid. The platform allows you to efficiently screen protocol literature, leveraging AI to pinpoint critical insights that can help you identify the most effective protocols for your specific research goals. By analyzing and comparing protocols from scientific literature, preprints, and patents, PubCompare.ai can highlight key differences in protocol effectiveness, enabling you to choose the best option for reproducibility and accuracy, and streamlining your iminodiacetic acid experiments.
In addition to its well-known uses as a chelating agent, iminodiacetic acid has some unique applications in specialized fields. For example, it can be used as a building block for the synthesis of certain pharmaceuticals and agrochemicals. Iminodiacetic acid derivatives have also been explored for their potential in biomedical applications, such as in the development of contrast agents for medical imaging.
More about "Iminodiacetic acid"
Iminodiacetic acid (IDA) is a versatile dicarboxylic amino acid with the chemical formula C4H7NO2.
It is widely used as a chelating agent in various industrial, research, and biochemical applications.
IDA can be employed to purify and immobilize proteins, enzymes, and other biomolecules, making it a valuable tool in biochemical research.
In addition to its chelating properties, IDA has a range of other uses.
It is commonly used in water treatment processes, metal extraction, and as a precursor for the synthesis of other organic compounds.
Researchers can optimize their IDA experiments with PubCompare.ai, an AI-driven comparison tool that helps identify the most reproducible and accurate protocols from scientific literature, preprints, and patents.
PubCompare.ai's intelligent algorithms analyze and compare protocols to find the best methods and products for your experiments, streamlining your research and helping you achieve better results.
The tool can also be used to explore related compounds, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), DGS-NTA-Ni, DOGS-NTA-Ni2+, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DGS-NTA, DOGS-NTA-Ni, HEPES, and NaCl, which are commonly used in biochemical and biophysical research.
By leveraging the insights and capabilities of PubCompare.ai, researchers can streamline their IDA-related experiments, optimize their protocols, and achieve more reliable and reproducible results, ultimately advancing their scientific discoveries.
The tool's user-friendly interface and powerful algorithms make it an invaluable resource for researchers working with IDA and related compounds.
