PBMC samples were transferred to a BSL-2 freezer and stored at − 80 °C until processed by iFASP [15 (link)]. Briefly, 30 µg of each inactivated PBMC lysate was added to 200 µL 8 M Urea/0.1 M Tris-HCl pH 8.5 and filtered through a Microcon-30 kDa Centrifugal Filter Unit with an Ultracel-30 membrane (Millipore; Cat# MRCF0R030) at 14,000×g for 15 min. Filters were washed 3× with 100 mM Tris pH 8.0, and proteins alkylated with 55 mM iodoacetamide followed by digestion with 4 µg Trypsin/Lys-C (Promega, Cat# V5071) overnight at 37 °C. TMT six-Plex labeling (Thermo Fisher Scientific; Cat# 90061) was performed directly on the FASP filters per the manufacturer’s instructions. TMT- labeled digests were then purified by C18 spin column, dried to completion by speed-vac and stored at − 20 °C until analyzed by LC–MS/MS.
Microcon 30 kda centrifugal filter unit
Manufactured by Merck Group
Sourced in United States, Germany
The Microcon-30 kDa centrifugal filter unit is a laboratory equipment designed for the concentration and purification of macromolecules, such as proteins, nucleic acids, or small molecules. The device utilizes centrifugal force to pass a sample solution through a semi-permeable membrane, allowing for the selective retention of molecules based on their molecular weight. The Microcon-30 kDa filter is capable of retaining molecules larger than 30 kilodaltons, making it a useful tool for sample preparation and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ultracel 30 membrane, by Merck Group (2 mentions)
Trypsin lys c, by Promega (2 mentions)
Trypsin, by Promega (1 mentions)
Tmt 6 plex labeling, by Thermo Fisher Scientific (1 mentions)
Oasis hlb cartridge, by Waters Corporation (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Allprep dna rna mirna universal kit, by Qiagen (1 mentions)
Paxgene blood mirna kit, by Qiagen (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Pbs buffer, by Merck Group (1 mentions)
Tris 2 carboxyethyl phosphine hydrochloride tcep hcl, by Thermo Fisher Scientific (1 mentions)
Chymotrypsin, by Thermo Fisher Scientific (1 mentions)
Diethyl pyrocarbonate, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Isopropanol, by Merck Group (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
10 protocols using microcon 30 kda centrifugal filter unit
1
Tandem Mass Tag Proteomics of PBMCs
2
Proteomic Sample Preparation via FASP
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Cells were harvested by counting each cell line in quadruplicate and were resuspended in a known volume of lysis buffer [8 M urea, 1% CHAPS, and 20 mM Hepes (pH 8.0)]. The suspension was pulsed six times with a sonic dismembrator on ice and was rotated end over end for 1 hour at 4°C. DNA was pelleted, and cell lysate was prepared for MS analysis.
Cell lysates were prepared using the filter-aided sample preparation (FASP) method. Briefly, 50 to 200 μg of protein per sample, at a concentration of 1 to 10 μg/μl, was reduced with DTT (final concentration of 3 mM) at 57°C for 1 hour and loaded onto a MicroCon 30-kDa centrifugal filter unit pre-equilibrated with 200 μl of FASP buffer A [8 M urea and 0.1 M tris-HCl (pH 7.8)] (Millipore). Following three washes with FASP buffer A, lysates were alkylated on a filter with 50 mM iodoacetamide for 45 min in the dark. Filter-bound lysates were then washed three times each with FASP buffer A and FASP buffer B [100 mM ammonium bicarbonate (pH 7.8)] and were digested overnight at room temperature with trypsin (Promega) at a 1:100 ratio of enzyme to protein. Peptides were eluted twice with 0.5 M NaCl. Tryptic peptides were desalted using an UltraMicro Spin Column, C18, (Harvard Apparatus), and the desalted peptide mixture was concentrated in a SpeedVac concentrator.
Cell lysates were prepared using the filter-aided sample preparation (FASP) method. Briefly, 50 to 200 μg of protein per sample, at a concentration of 1 to 10 μg/μl, was reduced with DTT (final concentration of 3 mM) at 57°C for 1 hour and loaded onto a MicroCon 30-kDa centrifugal filter unit pre-equilibrated with 200 μl of FASP buffer A [8 M urea and 0.1 M tris-HCl (pH 7.8)] (Millipore). Following three washes with FASP buffer A, lysates were alkylated on a filter with 50 mM iodoacetamide for 45 min in the dark. Filter-bound lysates were then washed three times each with FASP buffer A and FASP buffer B [100 mM ammonium bicarbonate (pH 7.8)] and were digested overnight at room temperature with trypsin (Promega) at a 1:100 ratio of enzyme to protein. Peptides were eluted twice with 0.5 M NaCl. Tryptic peptides were desalted using an UltraMicro Spin Column, C18, (Harvard Apparatus), and the desalted peptide mixture was concentrated in a SpeedVac concentrator.
3
Proteomic Analysis of Plasma Samples
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Plasma samples (6 time points/animal) were first processed in BSL-3 or BSL-4 containment by adding 25 µL SDS-PAGE solubilizing/reducing buffer to 75 µL sample and heating to 95 °C for 10 min. Samples were then removed from containment and stored at − 80 °C until processed by the iFASP method [28 (link)]. Briefly, 5 µL of each inactivated plasma sample was added to 200 µL 8 M Urea/100 mM Tris–HCL pH 8.5 (Solution UT8) and filtered through a Microcon-30 kDa Centrifugal Filter Unit with an Ultracel-30 membrane (Millipore, MRCF0R030) at 14,000 × G for 15 min. Following several washing steps with 100 mM Tris pH 8.0, proteins were alkylated with 55 mM Iodoacetamide and digested with 4 µg Trypsin/Lys-C (Promega, V5071) overnight at 37 °C. TMT 6-Plex labeling (Thermo Fisher, 90061) was performed directly on the FASP filters per the manufacturer’s instructions. All 6 single labelled samples were then combined at an equal volume, purified by C18 spin column, dried to completion by speed-vac and stored at − 20 °C until analyzed by LC MS/MS.
4
Protoplast Transformation and Gene Manipulation in Aspergillus
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The protoplast preparation and chemical-mediated transformation followed the method described by Dai et al. [58 (link)] for A. niger. Briefly, the 14.4 kb plasmid DNA of the β-alanine pathway transgene expression construct was linearized by restriction enzyme EcoRV and concentrated down to about 1 µg/µl with Microcon-30 kDa centrifugal filter unit (MilliporeSigma, Burlington, MA, USA). Ten microliters of the linearized plasmid DNA were used for protoplast transformation in A. niger. For transgene overexpression of A. niger aat1, pyc, the aat1-pyc, or mct1 gene in A. niger, about 3–5 µg of linearized plasmid DNAs by proper restriction enzymes were used for protoplast transformation. For the gene deletion construct of ald6a, ald6b, oah1, or uga2 gene homolog, about 1 µg of linearized plasmid DNAs by restriction enzyme PmeI was used for protoplast transformation in A. niger. Usually, about 5 to 12 transformed clones were picked randomly for the evaluation of 3-HP production and the effects of selected genes on 3-HP production. The chemical-mediated protoplast transformation of A. pseudoterreus was mainly followed the previous description [59 (link)].
5
Purification and Enrichment of MARylated Peptides
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Expression and purification of the eAf1521 macrodomain were done according to a published protocol (10 (link)). The peptide mixture was treated with PARG overnight (1.0 μg PARG per 1.0 mg peptide, Creative BioMart, Shirley, NY, Cat# PARG-31H) to obtain only MARylated peptides (2 (link)), and the peptides were enriched using the macrodomain affinity pull-down as described previously (10 (link)). Eighty percent of eluted ADPr peptides were processed using the MWCO filtration step using the Microcon-30 kDa Centrifugal Filter Unit, Millipore Sigma, Cat# MRCF0R03 (see eAf1521 Macrodomain Removal and Peptide Recovery ). The peptides were desalted using Oasis HLB cartridge (10 mg [Waters, 1 cc, Cat# 186008055] for the input peptides; 30 mg [Waters, 1 cc, Cat# WAT094225] for the MARylated peptides) by following its instruction and suspended in loading buffer (5.0% acetonitrile [Fisher Scientific, Cat# A955-1], 0.5% formic acid [Thermo Fisher Scientific, Cat# 28905] in water [Fisher Scientific, Cat# W6-1]) for LC-MS/MS analysis.
6
Efficient DNA Extraction from Whole Blood
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For separated cell types, DNA was extracted using an AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN). Whole blood DNA was isolated by combining the PAXgene Blood miRNA kit with the DNeasy Blood & Tissue Kit (QIAGEN). Briefly, samples were processed using the PAXgene Blood miRNA kit to the isopropanol precipitation step at which half the sample (~ 700 μl) was loaded onto a DNeasy Mini Spin column and processed using the manufacturer’s instructions for the DNeasy Blood & Tissue Kit followed by concentration with a Microcon-30 kDa Centrifugal Filter Unit (Millipore Sigma). DNA was quantified by a Qubit Fluorometer (ThermoFisher) and stored at − 20 °C.
7
Protein Sample Preparation Protocol
8
Extraction and Quantification of DNA from Urinary Sediment
9
In Vitro siRNA Release Kinetics
10
Cellular Uptake and Lactate Secretion Assay
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The workflow for accumulation experiments is shown in Figure 2 . Cells were seeded into 6-well plates two days prior, reaching 80–90% confluency on the day of the experiment. For the experiment, growth medium was withdrawn and cells were washed once with the respective medium, before fully supplemented MEM or glucose-reduced DMEM were added, containing 1MBq/mL 2-[18F]FDG (n = 5). Then, 1 h after addition of 2-[18F]FDG, aliquots of the supernatants were taken, cells were washed two times with PBS, detached with 500 µL of Accutase and resuspended in MEM. A 100 µL aliquot of the cell suspension was measured with a gamma counter (PerkinElmer, 2480 Automatic Gamma counter, Wizard23) and subsequently, cells were mixed with trypan blue and counted with a Neubauer chamber. Values are expressed as % applied dose (% AD) per 104 cells.
The aliquots of the supernatants were filtered through a Microcon-30 kDa Centrifugal Filter Unit (Merck KGaA, Darmstadt, Germany) to remove LDH from the medium. Then, lactate concentration was determined with a lactate assay kit (Merck KGaA, Darmstadt, Germany) following the supplier’s instructions.
The aliquots of the supernatants were filtered through a Microcon-30 kDa Centrifugal Filter Unit (Merck KGaA, Darmstadt, Germany) to remove LDH from the medium. Then, lactate concentration was determined with a lactate assay kit (Merck KGaA, Darmstadt, Germany) following the supplier’s instructions.
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