Initially, HEK293 cells were transfected with four plasmids encoding for the matuzumab heavy and light chains and cetuximab heavy and light chains. After five days of production, the culture supernatant was harvested and concentrated using a 10 kDa Amicon Ultra filter unit (EMD Millipore). The concentrated supernatant was then subjected to incubations using AM-VNAR1-functionalized magnetic beads according to the procedure described for the capturing assays in mouse serum. The eluates after the elution step were buffer-exchanged against PBS using Protein Desalting Spin colums (Thermo Fisher Scientific) and then applied to AC-VNAR2-coated beads. Incubation and elution was carried out as described above. The final eluates were subsequently concentrated using 10 kDa Amicon Ultra filter unit (EMD Millipore). After separation of the collected samples on a 15% SDS-PAGE, a silver staining was performed.
Amicon ultra filter unit
Manufactured by Merck Group
Sourced in United States, Germany
Amicon Ultra filter units are centrifugal devices used for the concentration and desalting of protein and other macromolecular solutions. They utilize a regenerated cellulose membrane with a specified molecular weight cutoff to retain the desired molecules while allowing smaller components to pass through.
Automatically generated - may contain errors
Lab products found in correlation
Magnetic bead, by Thermo Fisher Scientific (2 mentions)
Erms da470, by Merck Group (2 mentions)
Recombinant human gm csf, by Gentaur (2 mentions)
Protein a or protein g chromatography, by GE Healthcare (2 mentions)
Spectraplates, by PerkinElmer (2 mentions)
Ap conjugated anti human igg secondary antibody, by Southern Biotech (2 mentions)
Hitrap protein a hp column, by GE Healthcare (2 mentions)
Maxisorp plate, by Thermo Fisher Scientific (2 mentions)
Millex gp syringe filter unit, by Merck Group (2 mentions)
Stericup, by Merck Group (1 mentions)
Tskgel supersw3000 column, by Tosoh (1 mentions)
Protein quantification kit rapid, by Dojindo Laboratories (1 mentions)
Facsuite software, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Nickel nta agarose column, by Qiagen (1 mentions)
Cfx connect real time pcr instrument, by Bio-Rad (1 mentions)
Syto 82, by Thermo Fisher Scientific (1 mentions)
Victor nivo multimode plate reader, by PerkinElmer (1 mentions)
Prism 5, by GraphPad (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
27 protocols using amicon ultra filter unit
1
Dual-Antibody Production and Purification
2
Fc-fused anti-idiotypic vNAR Production
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Reformatting of the identified anti-idiotypic vNARs was accomplished upon genetically fusing them to an IgG1 Fc region encoded in the mammalian expression vector pEXPR-IBA42. VNAR variants were connected with the Fc region via a H20 hinge linker. Notably, the Fc sequence contained an Asn297Ala mutation in order to prevent glycosylation of the resulting fusion protein. Ligated constructs were sequence-verified and expressed in Expi293FTM cells subsequent to ExpiFectamineTM-mediated transient transfection according to the manufacturer’s instructions (Life Technologies). Culture supernatants were harvested, sterile-filtered (0.22 µm Stericup filter, EMD Millipore) and purified using ProSep® Protein A columns (EMD Millipore). Buffer exchange was performed with 10 kDa Amicon Ultra filter units (EMD Millipore). Analytical size exclusion chromatography was carried out using a TSKgel SuperSW3000 column (4.6 × 300 mm, Tosoh) at a flow rate of 0.35 mL/min.
3
Flow Cytometry Analysis of rCpEF1α Binding
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The purified rCpEF1α and rGST were concentrated by using Amicon Ultra filter units (50 K for rCpEF1α and 3 K for rGST) (Merck Millipore). The protein concentration was measured by using Protein Quantification Kit-Rapid (Dojindo laboratories, Kumamoto, Japan), and was diluted to 7.5 μM with elution buffer. Semi-confluent HCT-8 cells were washed twice, and treated with 500 μM EDTA in PBS for 5 min at 37 °C. Detached cells were washed with FACS buffer (PBS containing 2% fetal calf serum) by centrifugation at 1,500 rpm. Then 2 × 106 washed HCT-8 cells were incubated with 100 μL of 7.5 μM rCpEF1α or rGST for 2 h at 4 °C, and then washed twice with FACS buffer. The cells were then incubated with 100 μL of rabbit α-GST antibody (Sigma-Aldrich) at 1:1000 dilution for 30 min at 4 °C, washed with FACS buffer containing 2% FCS twice, incubated with 100 μL of Alexa 488-conjugated α-rabbit IgG goat antibody at 1:1000 dilution for 30 min at 4 °C, and washed with FACS buffer again. As a negative control, detached HCT-8 cells which were incubated with neither proteins nor antibodies were also prepared. The sample was then analyzed on BD FACSVerse (BD Bioscience) using BD FACSuite software (BD Biosciences). HCT-8 cells were gated on forward/side-light scatter to distinguish them from debris. Cells (10,000 events) were analysed by Alexa 488 channels.
4
SiMa Neuroblastoma Cell Culture
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SiMa cells (human neuroblastoma; DSMZ, Braunschweig, Germany; Marini et al. 1999 (link)) were maintained in RPMI medium supplemented with 10% FCS, glutamine, and penicillin/streptomycin, in an incubator at 37 °C and under a humidified atmosphere containing 5% CO2. Medium was exchanged every other day, and shortly before reaching confluency, cells were mechanically suspended and seeded at a lower density either on 12-mm glass cover slips (Menzel, Braunschweig, Germany) in a 24-well plastic multiwell plate (Sarstedt, Nümbrecht, Germany) for immunocytochemistry or on 6-well plastic multiwell plates (Sarstedt, Nümbrecht, Germany) for Western blot analysis. For antibody treatment, upon reaching the requested density, cells were pre-incubated for 4 h in an RPMI medium containing 1% FCS and subsequently for a time span as indicated in the text with 10 μg/ml of the respective antiserum. Prior to use, sodium azide of the applied antisera was removed by microdialysis with Amicon Ultra filter units (Merck, Darmstadt, Germany).
5
Purification of GABPA Protein from E. coli
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GABPA proteins were produced in the Six Pack E. coli cell line83 (link). Cells were suspended in buffer A (20 mM HEPES pH 8.0, 50 mM imidazole, 400 mM NaCl) supplemented with proteinase inhibitor cocktail (PIC), and sonicated. The cell extracts were loaded on Nickel-NTA agarose column (QiaGen, Hilden, Germany) and after washing, heterologous proteins were eluted in buffer B (20 mM HEPES pH 8.0, 250 mM imidazole, 400 mM NaCl). Samples were concentrated and the buffer was changed using Amicon ultra filter units (Merck, Darmstadt, Germany).
6
Monitoring RV-A2 RNA Accessibility via SYTO 82
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Ref. [48 (link)] was performed according to Real-Hohn et al. [49 (link)] with minor adaptations. RNA accessibility was monitored with SYTO 82 (Thermo Fisher, Waltham, MA, USA) in a Bio-Rad CFX Connect Real-Time PCR instrument (Hercules, CA, USA). Purified RV-A2 (~3.5 µg) in PBS minus PDS (control) and plus PDS at 200 µM final concentration, was incubated for 4 h at 4 °C (negligible virus breathing) or 34 °C (strong virus breathing). Unbound PDS was removed by ultrafiltration in 100 K Merck Amicon Ultra Filter units, followed by four PBS washes to eliminate the remaining unbound PDS. SYTO 82 was added to a final concentration of 5 µM, and the volumes were adjusted to 70 µL with PBS. Three 20 µL aliquots from each of these samples were dispensed into the wells of a thin-walled PCR plate, the temperature was ramped from 25–95 °C at 1.5 °C/min, and SYTO 82 light-up fluorescence was recorded. Six independent measurements were made for each condition. Data were rendered as a dot plot revealing the temperature at which the RNA becomes accessible for SYTO 82 binding [49 (link)].
7
Thioflavin T Fluorescence Assay for Ribooligonucleotides and Viral RNA
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Ribooligonucleotides were dissolved in 100 mM K+ or Na+ phosphate buffer (pH 7.4) to a final concentration of 5 µM, incubated for 10 min at 90 °C, followed by 10 min at 4 °C, and mixed with ThT to a final concentration of 5 µM. The samples were excited at 440 nm, and emission was measured at 490 nm using a PerkinElmer VICTOR Nivo Multimode Plate Reader (Waltham, MA, USA). The signal was acquired at 34 °C.
For the preparation of viral RNA cores (‘ex-virion RNA’), the protein shell of purified RV-A2 (~2 µg) was digested with 5 µg proteinase K overnight at 4 °C in a final volume of 10 µL. The ex-virion RNA samples were ultrafiltered using 100 K Merck Amicon Ultra Filter units according to the manufacturer’s protocol, followed by four 400 µL washes with 100 mM Na+ or K+ phosphate buffer (pH 7.4). The samples were mixed with ThT (final concentration of 5 µM), and the volume was adjusted to 100 µL with the respective buffers. The ThT fluorescence signal was acquired as described above, at 30 °C. The same samples were then incubated for 10 min at 60 °C followed by cooling to 30 °C over 30 min, and ThT fluorescence was again measured.
For the preparation of viral RNA cores (‘ex-virion RNA’), the protein shell of purified RV-A2 (~2 µg) was digested with 5 µg proteinase K overnight at 4 °C in a final volume of 10 µL. The ex-virion RNA samples were ultrafiltered using 100 K Merck Amicon Ultra Filter units according to the manufacturer’s protocol, followed by four 400 µL washes with 100 mM Na+ or K+ phosphate buffer (pH 7.4). The samples were mixed with ThT (final concentration of 5 µM), and the volume was adjusted to 100 µL with the respective buffers. The ThT fluorescence signal was acquired as described above, at 30 °C. The same samples were then incubated for 10 min at 60 °C followed by cooling to 30 °C over 30 min, and ThT fluorescence was again measured.
8
Purification of Recombinant Human Vimentin
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Recombinant human vimentin wild type (wt) and Cys328Ser in 8 M urea, 5 mM Tris-HCl pH 7.6, 1 mM EDTA, 10 mM 2-mercaptoethanol, 0.4 mM PMSF, and approximately 0.2 M KCl, purified essentially as described [57 (link)], were purchased from Biomedal (Spain). The proteins were ultrafiltrated using 10 K pore size Amicon Ultra filter units (Millipore) and step-wise dialyzed against 5 mM Pipes-Na pH 7.0, 1 mM DTT containing decreasing urea concentrations (8, 6, and 2 M). Final dialysis was performed for 16 h at 16 °C in 5 mM Pipes-Na, pH 7.0, 0.25 mM DTT [58 (link)]. Ultrafiltration was necessary for efficient removal of EDTA [58 (link)]. Protein concentration was estimated from its A280 nm, using an extinction coefficient of 22,450 M−1cm−1. Aliquots of the protein were kept at −80 °C until use.
9
Carbohydrate Modification in Echinococcus granulosus Protoscoleces
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Sodium metaperiodate-mediated modification of carbohydrates in EgPSC-ESPs was performed using a modification of Gómez-García et al. [23 (link)]. Briefly, 2 mg/ml of EgPSC-ESPs was incubated for a few seconds (v/v) with 50 mmol/l sodium acetate pH 4.5 at room temperature. The sample was divided into two to produce periodate-treated ESPs (P-ESPs) and mock periodate-treated ESPs (M-ESPs). Twenty millimolars of sodium metaperiodate (v/v) was added to P-ESPs, whereas the M-ESPs received acetate buffer without sodium metaperiodate. Both tubes were incubated for 30 min in the dark at room temperature with gentle shaking. The reaction was completed by further incubation with 100 mmol/l of sodium borohydride in PBS for 30 min at room temperature. Excess salt was removed by using Amicon Ultra Filter Units (Millipore, Billerica, MA, USA), and the protein concentration was determined.
10
Purification and Quantification of Anti-GM-CSF Antibodies
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Human mAbs and total IgG from PAP sera were purified by protein A or protein G chromatography (GE Healthcare). Total IgG from healthy donors were purified using the HiTrap Protein A HP columns (GE Healthcare) and concentrated using Amicon Ultra filter units (100 K, Millipore). Total GM-CSF antibodies were affinity-purified from PAP sera using magnetic beads (Invitrogen) conjugated with human GM-CSF. Total IgGs were quantified using ELISA plates coated with anti-human IgG (SouthernBiotech) using Certified Reference Material 470 (ERMs-DA470, Sigma-Aldrich) as standard. Binding to GM-CSF was tested by ELISA using 384-well SpectraPlates (PerkinElmer) for primary screenings or 96-well MaxiSorp plates (Nunc) for any following test. Briefly, ELISA plates were coated with 1 μg ml−1 of recombinant human GM-CSF (Gentaur), blocked with 1% BSA and incubated with titrated antibodies, followed by AP-conjugated anti-human IgG secondary antibodies (SouthernBiotech). Plates were then washed, substrate (p-NPP, Sigma) was added and plates were read at 405 nm. EC50 (ng ml−1) was calculated for every sample by nonlinear regression analysis using the GraphPad Prism 5 software.
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