As depicted in Fig 1 , the A, B, and C fractions as obtained above were re-dissolved in “Buffer A” (Agilent Technologies), and the non-fractioned reference plasma sample (P) was diluted. After centrifugation, these protein samples were immunodepleted of seven plasma HAPs (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, and fibrinogen) using a Multiple Affinity Removal Column Human-7 (4.6 mm inside diameter [ID]×50 mm; Agilent Technologies, USA) [1 (link)], according to the manufacturer’s instructions. “Buffer B” (Agilent Technologies) was used to elute the bound proteins. The flow-through fractions (LAPs and MAPs) were collected, desalted, and concentrated with 5-kDa Molecular Weight Cutoff Centrifugal filters (Amicon Ultra, Millipore) to generate the corresponding AF, BF, CF, and PF samples.
Buffer b
Manufactured by Agilent Technologies
Sourced in United States
Buffer B is a laboratory reagent used to maintain a specific pH range in various experimental procedures. It serves as a buffering solution to help stabilize the chemical environment and ensure consistent results.
Automatically generated - may contain errors
Lab products found in correlation
Oasis hlb cartridge, by Waters Corporation (2 mentions)
Trifluoroacetic acid uvasol, by Merck Group (2 mentions)
Multiple affinity removal system columns, by Agilent Technologies (2 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (2 mentions)
Amicon ultra, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Formic acid, by Avantor (1 mentions)
Milli q apparatus, by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Trypsin, by Promega (1 mentions)
Ultimate 3000 hplc system, by Thermo Fisher Scientific (1 mentions)
Mars hu6 column, by Agilent Technologies (1 mentions)
Formic acid, by BD (1 mentions)
Ch3cn, by BD (1 mentions)
9 protocols using buffer b
1
Immunodepletion of Plasma Proteins
2
Quantitative Proteomic Sample Preparation
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Iodoacetamide (IAA), tris(2-carboxyethyl) phosphine hydrochloride (TCEP), triethylammonium hydrogen carbonate buffer (TEAB) 1M pH = 8.5, sodium dodecyl sulfate (SDS), and LACB from bovine milk were purchased from Sigma (St. Louis, MO, USA). Formic acid (FA, 99%) was from BDH (VWR International Ltd., Poole, UK). Hydroxylamine solution 50 wt% in H2O (99.999%) was acquired from Aldrich (Milwaukee, WI, USA). Water (18.2 MΩ·cm at 25 °C) was obtained from a Milli-Q apparatus (Millipore, Billerica, MA, USA) and acetonitrile from BDH. Trifluoroacetic acid Uvasol® was sourced from Merck Millipore (Billerica, MA, USA). The sixplex tandem mass tags (TMTs) were purchased from Thermo Scientific (Rockford, IL, USA). Sequencing grade-modified trypsin was procured from Promega (Madison, WI, USA). For immuno-affinity depletion of 14 abundant human proteins, multiple affinity removal system (MARS) columns, Buffer A, and Buffer B were obtained from Agilent Technologies (Wilmington, DE, USA). Oasis HLB cartridges (1cc, 30 mg) were acquired from Waters (Milford, MA, USA) and strong cation-exchange (SCX) solid-phase extraction (SPE) cartridges from Phenomenex (Torrance, CA, USA).
3
Depletion of Abundant Serum Proteins
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Multiple affinity removal column (4.6×100 mm) was used to deplete the 14 most abundant proteins, namely albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, alpha 2-macroglobulin, alpha 1-acid glycoprotein, IgM, apolipoprotein AI, apolipoprotein AII, complement C3 and transthyretin. The affinity column was attached to the Dionex 3000 Ultimate RS LC system (Dionex, Sunnyvale, CA, USA) with buffer A (Agilent, Santa Clara, CA, USA) as mobile phase A and buffer B (Agilent, Santa Clara, CA, USA) as mobile phase B. LC gradient stated in the manufacture's handbook was used for the depletion. A 30 µL-aliquot of blood serum of each sample combined with 70 µL of buffer A was subjected to depletion separately. The buffer of depleted blood serum was exchanged into 50 mM ammonium bicarbonate (pH 8.0) using 5KDa MWCO 4 mL spin concentrator from Agilent (Santa Clara, CA, USA) following manufacture’s instruction. The depletion process was necessary to decrease the high dynamic range of serum proteome, thus allowing the identification and quantitation of low abundant proteins.
4
Plasma Protein Depletion Using MARS Hu-14
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Multiple Affinity Removal System (MARS Hu-14) cartridge was used for depletion of the samples. Prior to depletion, an aliquot of 10 µL of each plasma sample was diluted up to 200 µL with buffer A (Agilent Technologies, Palo Alto, CA, USA). The diluted samples were filtered through 0.22 μm cellulose acetate spin filters (Agilent Technologies) by centrifugation at 15000 × g for 2 min. Afterwards, an aliquot of 190 µL filtered and diluted plasma sample was loaded onto MARS Hu-14 cartridge and the flow-through (FT) fraction was collected by centrifugation for 2 min at 100 × g. Two successive wash steps with 400 µL of Buffer A were carried out to obtain maximum yield. The FT and wash (W) fractions were combined. The spin cartridge was washed with 2 ml of Buffer B (Agilent Technologies) to remove bound proteins and was then re-equilibrated with Buffer A. The remaining fractions (FT + W) were dried using a speed vac.
5
Serum depletion and SIS peptide analysis
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Trypsin was obtained from Promega (sequencing-grade modified, WI, USA). High-performance liquid chromatography (HPLC)-grade water and acetonitrile were purchased from Thermo Fisher Scientific (Bremen, Germany). Serum depletion was performed for the 6 most abundant proteins using a multiple affinity removal system (MARS), consisting of an LC column (Agilent, CA, USA); buffer A (Agilent, CA, USA) for sample loading, washes, and equilibration; and buffer B (Agilent, CA, USA) for elution. Unpurified stable isotope-labeled standard (SIS) peptides [isotopically labeled (13C and15N) amino acids] were obtained from JPT (Berlin, Germany) (30% to 70% purity, according to the manufacturer).
6
Abundant Protein Depletion from Synovial Fluid
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MARS-14 mini spin column (Agilent Technologies, Santa Clara, USA) was used to deplete top 14 abundant proteins (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM, apolipoprotein AI, apolipoprotein AII, complement C3, and transthyretin) from synovial fluid. Briefly, 20 µl synovial fluid was reconstituted in 140 µl of load/wash buffer (Buffer A, Agilent Technologies, Santa Clara, USA) and the loaded MARS spin column was then centrifuged at 2000 rpm for 10 min at room temperature. The flow through was collected separately and using elution buffer (Buffer B, Agilent Technologies, Santa Clara, USA), the bound high abundant proteins were eluted out. The entire protocol was followed as per the manufacturer’s instructions. A total of 5 mg equivalent protein was depleted to 250 µg, out of which 150 µg was separated on SDS-PAGE followed by in-gel trypsin digestion. Rest of 100 µg equivalent proteins was subjected to in-solution trypsin digestion followed by strong cation exchange chromatography (SCX)-based fractionation.
7
Depletion of Abundant Plasma Proteins
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Antibody affinity-based depletion of high abundance proteins present in human plasma was conducted using an Agilent Human top 14 Multiple Affinity Removal System (MARS) coupled to an Ultimate 3000 HPLC system (Thermo Scientific) following manufacturer’s instructions. Briefly, 80 µl plasma aliquots were centrifuged at 10,000×g for 10 min, diluted four times in Buffer A (Agilent Technologies, UK) and separated on the MARS column according to the manufacturer’s instructions. Protein depletion followed a sequence of isocratic elution steps: 100 % buffer A for 20 min at 0.125 ml/min followed by 0.7 ml/min for 2.5 min. Flow-through fractions containing the depleted plasma were collected between 7.5 and 14.5 min of each sample run. Between runs, the column was washed with buffer B (Agilent Technologies, UK) until the UV214nm trace was back to baseline. Each sample was injected four times to obtain sufficient quantity of protein for further analysis.
8
Serum Protein Depletion for Analysis
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Fifty µL serum samples from each subjects were diluted with commercially available Buffer A (Agilent Technologies, Wilmington, DE) at 1:5 ratio and centrifuged for 15 min at 15,000 g to remove particulate matter and lipids. Afterwards, 200 µL of the diluted sample was chromatographed through a Multiple Affinity Removal System (MARS-Hu6) Column (Agilent Technologies) at a flow rate of 0.5 ml/min. The flow through fraction embrace serum depleted the six most abundant proteins (albumin, alpha-1 antitrypsin, haptoglobin, transferring, IgA, and IgG) according to the manufacturer’s instructions and protocols. The bound proteins were eluted from the column with buffer B (Agilent Technologies). The abundant proteins depleted sera were collected and stored at −80 °C until time of analysis.
9
Quantitative Proteomics with TMT Labeling
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Iodoacetamide (IAA), tris(2‐carboxyethyl) phosphine hydrochloride (TCEP), triethylammonium hydrogen carbonate buffer 1 M pH 8.5, sodium dodecyl sulfate, and β‐lactoglobulin (LACB) from bovine milk were purchased from Sigma (St. Louis, MO, USA). Formic acid (FA, 99%) and CH3CN were from BDH (VWR International Ltd., Poole, UK). Hydroxylamine solution 50 wt% in H2O (99.999%) was acquired from Aldrich (Milwaukee, WI, USA). H2O (18.2 MΩ cm at 25°C) was obtained from a Milli‐Q apparatus (Millipore, Billerica, MA, USA). Trifluoroacetic acid Uvasol was sourced from Merck Millipore (Billerica, MA, USA). The 6‐plex isobaric tandem mass tags (TMTs) were purchased from Thermo Scientific (Rockford, IL, USA). Sequencing grade modified Lys‐C/trypsin was procured from Promega (Madison, WI, USA). For immuno‐affinity depletion of 14 abundant human proteins, multiple affinity removal system (MARS) columns, Buffer A, and Buffer B were obtained from Agilent Technologies (Wilmington, DE, USA). Oasis HLB cartridges (1cc, 30 mg) were acquired from Waters (Milford, MA, USA) and Strata‐X 33u Polymeric reversed‐phase (RP) and Strata‐X‐C 33u Polymeric strong cation‐exchange (SCX) solid‐phase extraction (SPE) cartridges (30 mg/1 mL) from Phenomenex (Torrance, CA, USA).
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