For purification of Trento and Z AAT from cell media, HEK293T cells were transfected with poly(ethylenimine) and incubated in serum‐free DMEM for 36 h. The conditioned medium was collected, centrifuged for 5 min at 3000 g , and AAT was purified by affinity chromatography on Alpha‐1 Antitrypsin Select (GE Healthcare). About 36 mL of cell supernatant was loaded onto 500 μL of matrix, washed with 150 mm NaCl/20 mm Tris pH 7.4 and eluted with 1 mL of 2 m MgCl2/20 mm Tris pH 7.4. The purified samples were desalted by Amicon Ultra‐0.5 centrifugal Filter devices (Millipore, Merck, Milan, Italy). A similar protocol was used to purify S and Z from plasma diluted 1 : 5 with 150 mm NaCl/20 mm Tris pH 7.4. Polymerization of purified proteins at 1–5 μm concentration in phosphate buffer pH 7.4 was induced either by heating at 55 °C for 4 h or by treating for 30 h at room temperature with 3 m GuHCl or 3 m urea.
Amicon ultra 0.5 centrifugal filter device
Manufactured by Merck Group
Sourced in Germany, United States
The Amicon Ultra-0.5 Centrifugal Filter Devices are laboratory equipment used for sample concentration and buffer exchange. They utilize a centrifugal force to facilitate the separation of macromolecules, such as proteins, from smaller molecules or solvents. The devices feature a semi-permeable membrane that allows the passage of smaller molecules while retaining the larger target molecules.
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Lab products found in correlation
Quantikine elisa hyaluronan immunoassay, by R&D Systems (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Superase inhibitor, by Thermo Fisher Scientific (2 mentions)
Poly 1 c, by Merck Group (2 mentions)
Chemidoc mp system, by Bio-Rad (2 mentions)
Alpha 1 antitrypsin select, by GE Healthcare (1 mentions)
N ethylmaleimide, by Merck Group (1 mentions)
Dcp bio1, by Merck Group (1 mentions)
Catalase, by Worthington (1 mentions)
Dcp bio1, by Kerafast (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Exoquick exosome precipitation solution, by System Biosciences (1 mentions)
Amicon ultra 100k device, by Merck Group (1 mentions)
Luminex 100, by Bio-Rad (1 mentions)
Complete mini, by Roche (1 mentions)
Quantichrom arginase assay kit, by BioAssay Systems (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Transdux, by System Biosciences (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Nupage bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
47 protocols using amicon ultra 0.5 centrifugal filter device
1
Purification and Polymerization of Alpha-1 Antitrypsin Variants
2
Profiling Protein Sulfenylation and Perthiosulfenylation
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For analysis of protein sulfenylation (Cys-SOH) and perthiosulfenylation (Cys-SSOH), cells were lysed in Western solubilization buffer (WSB) containing 1 mM DCP-bio1 (Kerafast or EMD Millipore Sigma), 200 U/mL catalase (Worthington, Lakewood, NJ) and 10 mM N-ethylmaleimide (Sigma) and incubated for 1 h on ice. Equal amounts of cell lysates were mixed with Laemli sample buffer, either in the presence or absence of 25 mmol/L DTT for 30 min, and separated by 10% SDS-PAGE for Western blotting with streptavidin-peroxidase (see below). For analysis of specific proteins of interest, excess DCP-bio1 reagent was removed from DCP-bio1-derivatized lysates by 6 successive washes with 20 mM Tris-HCl (pH 7.4) on Amicon Ultra-0.5 Centrifugal Filter Devices (Millipore). DCP-bio1-tagged proteins were subsequently collected with high capacity NeutrAvidin-agarose beads (Pierce) and washed successively with 1% SDS, 4 mol/L urea, and 1 mol/L NaCl [28] (link). Beads were then washed with 100 mmol/L ammonium bicarbonate either in the presence or absence of 10 mmol/L DTT for 30 min to assess the difference of sulfenic acid and perthiosulfenic acid tagged proteins.
3
Small RNA Extraction and Quantification
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Total smRNAs were extracted from ceca mucus (n = 4–5 per group) following mirVanaTM miRNA isolation kit (Ambion®) following the manufacturer’s procedure for smRNA purification. Subsequently, smRNAs were further purified using Amicon® Ultra-0.5 Centrifugal Filter Devices (Millipore) as described previously (Liu et al., 2016 (link)). As calculated by NanoDrop 2000, the A260/A280 ratios for all extracts were approximately 2.0, indicating an acceptable purity of the smRNAs. smRNA quantity was calculated via QubitTM microRNA Assay Kit (which broadly detects small RNA molecules), and concentrations were calculated by adjusting for the weight (g) of each sample.
4
Isolation and Characterization of Extracellular Vesicles
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Samples were centrifuged at 350 × g for 10 min at 4 °C to remove cell debris, then filtered through a 0.22 μm filter. For EVs characteristics analysis and functional assays, 2.5 ml of samples were diluted with 7.5 ml of PBS and concentrated using Amicon ultra-0.5 centrifugal filter devices (Millipore, Amicon Ultra 100 K device) at 3000 × g for 30 min at 4 °C. One hundred µl retentate was diluted with 1.4 ml of PBS and subjected to centrifugation at 10,000 × g for 30 min at 4 °C. The pellets were resuspended in 1.5 ml PBS and ultracentrifuged at 120,000 × g for 90 min at 4 °C. The pellets were resuspended in 50 µl PBS and stored at -80 °C. For quantitative reverse transcription PCR (QRT-PCR) validation analysis, the plasma–derived EVs were exacted by ExoQuick exosome precipitation solution (System Biosciences, USA) according to the manufacturer’s instructions. The purified EVs were confirmed using transmission electron microscope images analysis, the nanoparticle tracking assay, immunoblotting and flow cytometry, respectively. The EVs were quantified using a direct ELISA-based method to quantify the EVs surface marker CD63 according to the manufacturer’s instructions (System Biosciences, USA).
5
Quantifying Secreted Immune Factors in BAL
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Concentrations of secreted cytokines and chemokines MCP1 (CCL2), KC (CXCL1), MIP-1α (CCL3), IL-6 and TNF-α in BAL were determined using a magnetic bead-based MILLIPLEX MAG multiplex assay (Millipore) and analyzed on a Luminex100 (BIO-RAD, Munich, Germany). For this assay, BAL fluid was concentrated (10×) by ultrafiltration in Amicon Ultra-0.5 centrifugal filter devices (Millipore).
6
Purification and Delivery of miRNA Inhibitors
7
Quantifying Arginase-1 in Prostate Cancer MDSCs
8
IBTK Phosphorylation by mTOR/S6K1
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The pGEX-4T-2 construct containing IBTK900-1150aa was transformed into Escherichia coli Rosetta (DE3) to express the recombinant GST-tagged proteins. These proteins underwent a two-step purification process on glutathione-agarose beads (Pharmacia), followed by affinity purification on Amicon Ultra-0.5 Centrifugal Filter Devices (Millipore). IBTK phosphorylation levels were assessed using in vitro kinase assays. Purified GST-tagged IBTK900-1150aa at 2 μg was mixed with kinases mTOR/mLST8 or S6K1 (Carna Biosciences) in a reaction mixture that contained 10× kinase buffer (CST), 10 mM ATP (Sigma) for 1 hr at 30°C. The reaction was terminated by adding 2× SDS loading buffer and boiling at 105°C for 5 min. SDS-PAGE was used to separate proteins, with gel bands cut out and subjected to MS sequencing.
9
Genome-wide siRNA Screening to Identify Radiation-Modulating Genes
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The negative screening was performed using the Decode RNAi Annotated Genome Screening Library: Negative Selection Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. A549 cells were infected with a lentiviral siRNA expression library using TransDux (System Biosciences, Palo Alto, CA, USA) (pool 1 and pool2). Two days after infection, noninfected cells were removed by three-day treatment with puromycin, and cells were divided into two groups, one of which was irradiated with 4 Gy γ-ray, the other was stored as non-irradiated control. Seven days after irradiation, genomic DNA was purified using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Barcode sequences incorporated into genomic DNA were amplified by PCR using primers included in the kit and labeled with cyanine-3 for non-irradiated control, with cyanine-5 for the irradiated group. After purification with Amicon Ultra-0.5 Centrifugal Filter Devices (Millipore, Burlington, MA, USA), the labeled sequences were hybridized with a microarray slide for 17 h at 65 °C in ICES/NMB-001 (Agilent Technologies, Santa Clara, CA, USA). Signals from cyanine-3 or cyanine-5 were measured with DNA Microarray Scanner/Agilent Scan Control Software (Agilent Technologies).
10
Reversibility of BaPif1 Inhibition
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The reversibility
of BaPif1 inhibition was tested by filtration through Amicon Ultra-0.5
centrifugal filter devices (Millipore) with a cut-off of 10 kDa followed
by dilution of the concentrated retentate. Samples of 0.1 mg/mL BaPif1
in the assay buffer described above were incubated with 5% DMSO or
with a 50 μM concentration of the tested compounds in 5% DMSO
for 30 min and then filtered by centrifugation according to the manufacturer’s
instructions. The retentate sample was diluted 10-fold with assay
buffer to recover the original volume. This procedure was repeated
three times (total dilution was 1000-fold), and finally, the diluted
retentate was used to measure the BaPif1 ATPase activity as described
above.
of BaPif1 inhibition was tested by filtration through Amicon Ultra-0.5
centrifugal filter devices (Millipore) with a cut-off of 10 kDa followed
by dilution of the concentrated retentate. Samples of 0.1 mg/mL BaPif1
in the assay buffer described above were incubated with 5% DMSO or
with a 50 μM concentration of the tested compounds in 5% DMSO
for 30 min and then filtered by centrifugation according to the manufacturer’s
instructions. The retentate sample was diluted 10-fold with assay
buffer to recover the original volume. This procedure was repeated
three times (total dilution was 1000-fold), and finally, the diluted
retentate was used to measure the BaPif1 ATPase activity as described
above.
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