The media were centrifuged at 20,400 × g for 5 min and filtered through 30 kDa Amicon ultra and washed 13 times with PBS to eliminate the compounds. The media were then separated by a Superose six column (GE healthcare) in PBS at 0.5 ml/min with an AKTA explorer 10S (GE healthcare). HEK293 cells expressing P301S tau with RD transduction were sonicated with ice cold PBS supplemented with complete and PhosSTOP. After centrifugation at 100,000 × g for 20 min, the supernatants were collected as PBS-soluble cell extracts and loaded onto the column. Recombinant 2N4R tau (2.1 μg) was loaded as a control. HMW calibration kit (GE healthcare) was used to estimate molecular weights.
Akta explorer 10
Manufactured by GE Healthcare
Sourced in United Kingdom, United States, Sweden
The AKTA Explorer 10 is a versatile liquid chromatography system designed for protein purification. It offers automated control and monitoring of various parameters such as flow rate, pressure, and UV absorbance during the purification process. The system is intended for use in research and development laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Superdex 200 10 300 gl column, by GE Healthcare (2 mentions)
Hmw calibration kit, by GE Healthcare (1 mentions)
Superose six column, by GE Healthcare (1 mentions)
Mouse monoclonal antibody isotyping test kit, by Bio-Rad (1 mentions)
Pgex 6p 3 vector, by GE Healthcare (1 mentions)
Glutathione sepharose, by GE Healthcare (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Superdex 75 column, by GE Healthcare (1 mentions)
Superdex 200 10 300 gl, by GE Healthcare (1 mentions)
Superdex 200 hr 10 30, by GE Healthcare (1 mentions)
Er2566 cells, by New England Biolabs (1 mentions)
Pefabloc sc, by Roche (1 mentions)
Sonifier 250d, by Emerson (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
0.20 mm pore filter, by Merck Group (1 mentions)
Hiprep 26 10, by GE Healthcare (1 mentions)
Mabselect sure, by GE Healthcare (1 mentions)
Capto q, by GE Healthcare (1 mentions)
15 protocols using akta explorer 10
1
Purification and Characterization of Tau Aggregates
2
Generating Anti-Monkey Podoplanin Antibodies
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A monkey podoplanin cDNA region encoding amino acids 76 to 89 (226–267 bp) was tandemly connected 21 times in a pGEX-6P-3 vector (GE Healthcare), replicated in BL21 (DE3) E. coli as the GST-tagged monkey podoplanin peptide (76–89 aa), and purified using glutathione sepharose (GE Healthcare). Six-week-old female BALB/c mice were immunized by neck and ventral subcutaneous injections of the GST-tagged peptide with Titer MAX Gold adjuvant (Titer MAX, Norcross, GA, USA). Cell fusion was performed by the procedure described previously [14 (link)]. Positive clones were subcloned three times by limiting dilution, and some were purified from ascites as described previously [48 (link)]. For the acute toxicity test with cynomolgus monkeys, large-scale purification of control IgG1 and 2F7 antibody was conducted. Six-week-old female BALB/c-nu/nu mice were injected intraperitoneally with 2F7-secreting hybridomas or control IgG1-secreting hybridomas (DIG104.10H.1) suspended in Hanks’ Balanced Salt Solution (HBSS, Gibco). Ascitic fluid was collected for purification of the antibody by salting out with ammonium sulfate and performing affinity column chromatography with protein G using AKTA Explorer 10S (GE Healthcare). The IgG isotype was determined with a Mouse Monoclonal Antibody Isotyping Test Kit (AbD Serotec, Oxford, UK).
3
Purification and Quantification of α-Synuclein
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ISF samples were pooled and separated by a size exclusion chromatography using Superdex 75 column (GE healthcare). PBS was run at 0.5 ml/min with an AKTA explorer 10S (GE healthcare). α-Synuclein levels in each fraction were determined α-synuclein ELISA. LMW calibration kit to determine molecular weights was purchased from GE healthcare.
4
LUBAC Isolation from Liver Tissue
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Liver sections were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 1 mM DTT, and 1 mM PMSF, protease inhibitor cocktail). Since the LUBAC is located in the cytosolic fraction, as reported previously, detergent is not required [14 (link)]. After adding an equal volume of detergent-free lysis buffer containing 300 mM NaCl, lysates were centrifuged at 100,000 g for 30 min. The supernatants were then applied to gel filtration using a Superdex 200 10/300 GL (GE Healthcare) and fractionated at 1 mL/min in elution buffer containing 50 mM Tris-HCl pH 7.5 and 150 mM NaCl using an AKTA explorer 10S (GE Healthcare). The collected samples were subjected to immunoblotting analysis.
5
Separation of Serum Macro and Free TSH
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To separate macro TSH from free TSH in the serum samples, gel filtration chromatography was performed using a Low/High Molecular Weight Kit (GE Healthcare, Japan). Pooled serum samples from 10 participants were passed through a 0.22 μm PES syringe filter (Starna Scientific, UK), then injected into a Superdex 200HR10/30 (GE Healthcare), which was equilibrated with 0.05 M of phosphate buffer in 0.15 M of NaCl solution adjusted to pH 7.0 and run at a flow-rate of 0.5 ml/minute on an AKTA Explorer 10 s (GE Healthcare). Chromatography was monitored using Prime View UNICON 5.31 software.
6
Recombinant Rif1 Protein Expression and Antibody Generation
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Recombinant proteins, mouse Rif1 N206 aa or Rif1 UCRIII (the upstream segments of C-terminal conserved region III (24 (link)); 1673–1851 aa), were expressed in ER2566 cells (New England Biolabs) transformed with pGEX-4T1-N206 or pGEX-4T1-UCRIII by induction with 0.1 mM IPTG at 30 °C for 3 h. The proteins were solubilized by sonication of cells using Sonifier 250D (Branson) on ice in PBS supplemented with 1% Triton X-100, protease inhibitors (1 μg/ml pepstatin A, 1.5 μg/ml aprotinin, 1 μg/ml leupeptin (Sigma-Aldrich), 0.4 mM Pefabloc SC (Roche)), and 5 mM DTT. After removal of insoluble materials by centrifugation at 60,000g for 20 min at 4 °C, GST-fused proteins were purified using glutathione Sepharose 4B (GE Healthcare). For generation of polyclonal antibodies, 0.3 mg of purified proteins was used to immunize a rabbit each time for total five times, and two animals for each antibody were used (Bio Regenerations). The antibodies were affinity-purified by AKTA Explorer 10S (GE Healthcare) using recombinant antigen protein-conjugated HiTrap NHS-activated HP columns following manufacturer’s instructions and validated the specificities. Other antibodies used in this study are listed in Table S2 .
7
Size-Exclusion Chromatography of Fluorescent Proteins
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Size-exclusion chromatography was performed using an A ¨KTA explorer 10S equipped with a Superdex 200 10/300 GL column (GE Healthcare), where 500 ml of purified FPs were loaded on the column after dilution in 20 mM HEPES (pH 7.4) containing 150 mM NaCl to the desired concentration and filtration through a 0.20 mm pore filter (Millipore). The same buffer was used for the mobile phase at a flow rate of 0.75 ml/min. Absorption was measured at the wavelength of the absorption spectrum peak.
8
Purification of Elastin-Like Polypeptides
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The protocol followed is similar to the one described in 2.4.3, with the difference that ammonium sulfate was used for the precipitation of the ELPs during the ITC cycles and DMSO was employed to aid their resolubilization. In particular, the ELP-containing supernatant was supplemented with 10% v/v saturated ammonium sulfate solution to aggregate the ELPs at room temperature. The first hot spin was conducted by centrifugation at 10,000 rpm, 15 min, 25 °C, and the ELP pellets were resolubilized in DMSO after a freeze–thaw cycle. The mixture was diluted with cold 15 mM phosphate 15 mM NaCl buffer pH 7.4 (final content of DMSO 10% v/v) before proceeding to the first cold spin at 15,000 rpm, 10 min, 4 °C. The ITC cycles were repeated another 3 times without the use of DMSO. For each cold spin, the volume of buffer to resolubilize the ELPs was reduced to half, and thus for every hot spin the volume of ammonium sulfate was also reduced to half. The supernatant isolated after cold spin 4 was desalted on a HiPrep 26/10 (GE Healthcare Life Sciences) with an AKTA Explorer 10 (GE Healthcare Life Sciences) at 1 mL/min ultrapure water.
9
High-Yield Antibody Purification Protocol
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Stable cell lines with high antibody expression levels were obtained through methotrexate pressurization. The cell line with the highest antibody expression level was cultured in a 5-L bioreactor (Z310110010, Applikon, Delft, Netherlands). The cell culture supernatant was concentrated and purified using a purification system (AKTA Explorer 10, General Electric Company, Boston, Massachusetts, USA) and a three-step chromatographic method [26 ] as follows: MabSelect SuRe™ (17,543,802, General Electric Company) affinity chromatography to bind antibodies in the cell culture supernatant; Capto™ S (17,544,101, General Electric Company) cation exchange chromatography to exchange target antibodies by conferring a positive charge according to the isoelectric point of antibody; and Capto™ Q (17,531,602, General Electric Company) anion exchange chromatography to remove endotoxins and nucleic acids from the antibodies.
10
Purification and Kinetic Analysis of AHAIR
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Wild-type and mutant AHAIR were purified from BL21/pET28a-ilvC and BL21/pET28a-ilvCTM, and all purification steps were performed at 4 °C. Cultivated cells were disrupted by sonication and centrifuged to remove cell debris. Then, extract was purified by a protein purifier (AKTA Explorer 10, GE Healthcare, Uppsala, Sweden). Purified wild-type AHAIR and mutant AHAIR were used to perform enzyme activity for kinetic analysis. The decrease of NAD(P)H was measured at 340 nm. The α-acetolactate and NAD(P)H concentrations were from 0 to 20 mmol L−1 and 0 to 1 mmol L−1, respectively.
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