The plasma pools were depleted of the proteins by using the Multiple Affinity Removal LC Column-Human 14 (Cat. 5188-6560, Agilent, CA, USA) according to the protocol from the manufacturer. The desalination and concentration of the low-abundance components were performed by using a 10 kDa ultrafiltration tube. One volume of SDT (4% SDS, 100 mM Tris/HCl pH 7.6, 0.1 M dithiothreitol) buffer was added, and then boiled for 15 min, then centrifuged for 20 min with 14,000×g. The BCA Protein Assay Kit (Bio-Rad, Hercules, CA, USA) was used for protein quantification, and all the supernatant samples were stored at −80 °C for further measurement.
Multiple affinity removal lc column human 14
Manufactured by Agilent Technologies
Sourced in United States
The Agilent Multiple Affinity Removal LC Column-Human 14 is a chromatography column designed for the removal of high-abundance proteins from human serum or plasma samples. The column uses a proprietary resin to selectively deplete 14 high-abundance proteins, allowing for the enrichment and analysis of lower-abundance proteins.
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Lab products found in correlation
Bca protein assay kit, by Bio-Rad (1 mentions)
Agilent 1260 infinity 2 hplc system, by Agilent Technologies (1 mentions)
Easy nlc, by Thermo Fisher Scientific (1 mentions)
Itraq kit, by AB Sciex (1 mentions)
Akta purifier 100 system, by GE Healthcare (1 mentions)
Agilent 1100, by Agilent Technologies (1 mentions)
6 protocols using multiple affinity removal lc column human 14
1
Depleting Abundant Plasma Proteins
2
Quantitative Proteome Analysis of Alzheimer's Disease
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Equal volume of serum samples from five patients in each group were pooled for iTRAQ analysis. The multiple affinity removal LC column – human 14 (Agilent Technologies) was used to remove the high abundance proteins from the samples, accomplishing by ultrafiltration and concentration in the 10 kDa ultrafiltration tubes. Peptide mixtures (100 μg) from each group were labeled using the iTRAQ Reagent‐plex Multiplex Kit (AB SCIEX) following manufacturer's protocol. The digested peptides were labeled with six iTRAQ tags (reagent 115:normal 1; reagent 118:AD1; reagent 116:normal 2; reagent 119:AD 2; reagent 117:normal 3; reagent 121:AD 3). The labeled peptides of each group were mixed and graded by Agilent 1260 infinity II HPLC system. Then liquid chromatography‐mass spectrometry/mass spectrometry (LC–MS/MS) analysis was performed on Easy nLC (Thermo Scientific).
3
Serum Protein Depletion and Concentration
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An Agilent Multiple Affinity Removal LC Column (Human 14) (Agilent, CA, USA) was used to remove high-abundance proteins in accordance with the protocol to obtain a low-abundance component solution in the serum sample. A 5 kD ultrafiltration tube was used for ultrafiltration and concentration, and one-fold volume of SDT lysis was added into the system, which was incubated in a water bath at 100 °C for 10 min and centrifuged at 14,000 × g for 15 min. The supernatant was extracted for protein quantification using a BCA kit, and the samples were subpackaged and stored at −80 °C.
4
iTRAQ-based Serum Proteome Analysis
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Equal amounts of serum from each sample were acquired and pooled. Multiple Affinity Removal LC Column-Human 14 (Agilent) was employed to remove high abundance serum proteins. Bradford assay was performed to determine the concentrations of proteins. Then, proteins were digested into peptides with trypsin and desalted with C18-SD Extraction Disk Cartridge (66872-U sigma). Peptides were labeled with reagents following the iTRAQ kit (AB SCIEX) instructions and then mixed. For LC-MS/MS analysis, the complexity of peptides sample was firstly reduced utilizing strong cation exchange (SCX) chromatography. The labeled peptides were separated using a Polysulfoethyl column (4.6 × 100 mm, 5 um, 200 Å, PolyLCInc, Maryland, USA) on the AKTA Purifier 100 system (GE Healthcare). The peptide fractions were collected at an interval of one minute and grouped into 12 components according to SCX chromatograms. Components were lyophilized in a vacuum concentrator and desalted using C18 columns.
5
Quantitative Proteomic Analysis of Biological Samples
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Agilent multiple affinity removal LC column-human 14 of the corresponding samples was used to remove the high abundance protein, and the low abundance component solution was obtained. Mass spectrometry was performed after FASP digestion, and each sample was separated by a nanoliter flow rate Easy nLC system. Buffer A solution was 0.1% formic acid aqueous solution and B solution was 0.1% formic acid acetonitrile aqueous solution (acetonitrile is 80%). The chromatographic column was equilibrated with 100% liquid A, and the samples were separated from the automatic sampler to the analytical column (Thermo scientific, Acclaim PepMap RSLC 50 µm × 15 cm, nano viper, P/N164943) at a flow rate of 300 nl/min. After chromatographic separation was carried out, samples were analyzed by a Q-Exactive Plus spectrometer. The analysis time was 60 min (Zhang et al., 2016 (link)).
6
Immunodepletion and Preprocessing of Serum Proteins
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Sera were immunodepleted at Biosystems International Ltd., (Debrecen, Hungary) for 14 highly abundant serum proteins on an Agilent 1100 HPLC system using Multiple Affinity Removal LC Column-Human 14 (Agilent Technologies) according to the manufacturer’s protocol. To improve the resolution of 2D gels, immunodepleted serum samples were lyophilized, delipidated, and salt-depleted at the Proteomics Laboratory of the Eotvos Lorand University (Budapest, Hungary) (339 (link)). The delipidated and salt-depleted plasma protein samples were dissolved in lysis buffer, and their pH was adjusted to 8.0.
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