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Ammonium bicarbonate abc

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Ammonium bicarbonate (ABC) is a chemical compound with the formula (NH4)HCO3. It is a white, crystalline solid that is commonly used in various laboratory applications. ABC serves as a pH buffer, maintaining a slightly alkaline environment. It is also used as a leavening agent in baking and as an effervescent agent in certain pharmaceutical and personal care products.

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Lab products found in correlation

Dithiothreitol (dtt), by Merck Group (8 mentions) Trifluoroacetic acid, by Merck Group (3 mentions) Sequencing grade modified trypsin, by Promega (3 mentions) Iodoacetamide, by Merck Group (3 mentions) Urea, by Merck Group (3 mentions) Formic acid, by Merck Group (3 mentions) Iodoacetamide iaa, by Merck Group (3 mentions) Trypsin, by Merck Group (2 mentions) Urea, by GE Healthcare (2 mentions) Iodoacetamide iam, by Merck Group (2 mentions) Rg503h, by Merck Group (2 mentions) Dir dye, by Thermo Fisher Scientific (2 mentions) Polyvinyl alcohol (pva), by Thermo Fisher Scientific (2 mentions) Polyvinyl alcohol (pva), by Merck Group (2 mentions) Dithiothreitol (dtt), by Bio-Rad (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Trypsin lysc mixture, by Promega (2 mentions) Trypsin gold, by Promega (2 mentions) Sep pak c18 96 well plate, by Waters Corporation (1 mentions) Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)

21 protocols using ammonium bicarbonate abc

1

In-Solution Tryptic Digestion and Cleanup

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In-solution tryptic digestion and peptide cleanup were simultaneously performed in a 96-well plate for high reproducibility. Each depleted sample was supplemented with 8 M urea in 100 mM ammonium bicarbonate (ABC) (Sigma) and incubated at room temperature for 20 min. The samples were homogenized by vortexing and sonication twice. To each sample, dithiothreitol (Sigma) was added to be 10 mM for protein reduction at RT for 1 h. Then, iodoacetamide (Sigma) was added to be 30 mM for the cysteine alkylation at RT for 30 min in the dark. Samples were then diluted with 100 mM ABC prior to the addition of trypsin (MS grade, Pierce) at 1:50 of trypsin:sample ratio (w/w), and incubated at 37 °C for overnight. The trypsin was inactivated by acidification with 0.4% trifluoroacetic acid (Sigma). The acidified digests were immediately processed using a Sep-Pak C18 96-well plate (100 mg C18 sorbent per well, Waters). The peptides were eluted with 80% acetonitrile and then dried in a vacuum centrifuge.
Lee J.H., Jung J.H., Kim J., Baek W.K., Rhee J., Kim T.H., Kim S.H., Kim K.P., Son C.N, & Kim J.S. (2020). Proteomic analysis of human synovial fluid reveals potential diagnostic biomarkers for ankylosing spondylitis. Clinical Proteomics, 17, 20.
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2

Membrane Protein Purification and Analysis

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Nylon membranes (LoProdyne LP, pore size 1.2 μm, 110 μm thickness) were acquired from Pall Corporation (Port Washington, New York). Poly(sodium 4-styrenesulfonate) (Mw ~ 70,000) (PSS), sodium chloride, trypsin (from porcine pancreas type IX-S, lypholized powder), hydrochloric acid, poly(acrylic acid) solution (Mw ~ 100,000), branched polyethylenimine (Mw ~ 25,000) , iodacetamide, acetonitrile, ammonium bicarbonate (ABC), and sodium dodecyl sulphate (BioReagent, suitable for electrophoresis) were purchased from Sigma Aldrich (St. Louis, Missouri). Sequencing grade modified trypsin was obtained from Promega (Madison, Wisconsin), and Mini-Protean 4-20% TGX precast gels were purchased from Bio Rad (Hercules, California). HiPPR Detergent Removal columns (0.1 mL), dithiothreitol (DTT, molecular biology grade), unstained protein molecular weight marker, extra thick western blotting filter paper, and Pierce C-18 Spin Columns, were acquired from Thermo Scientific (Waltham, Massachusetts). Methanol, glycine, and ultra-pure Tris were purchased from VWR (Radnor, Pennsylvania), and low fluorescence PVDF (0.45 μm) was obtained from Azure Biosystems (Dublin, California). Zwittergent 3-16 detergent was purchased from EMD Millipore (Burlington, Massachusetts).
Bickner A.N., Champion M.M., Hummon A.B, & Bruening M.L. (2020). Electroblotting through a tryptic membrane for LC-MS/MS analysis of proteins separated in electrophoretic gels. The Analyst, 145(23), 7724-7735.
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3

Proteomic Profiling of Thermal Stress

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For the analysis of the proteomic profile at 20°C or 37°C, protein extracts were fractionated by SDS-PAGE (in a 10% SDS-polyacrylamide gel) till the whole proteome had penetrated in the resolving gel (about 1 cm of total migration). Gels were stained with Colloidal Blue Staining Kit (Invitrogen). Each proteome was excised and cut into small pieces prior to manual in-gel digestion with trypsin. Excised bands were separately detained with 50 mM ammonium bicarbonate (ABC) (Sigma-Aldrich) and 50% acetonitrile (ACN) (Fisher Chemical). Samples were then reduced with 10 mM dithiothreitol (Bio-Rad) in 50 mM ABC and alkylated with 55 mM iodoacetamide (GE Healthcare Life Sciences) in 50 mM ABC. Then, gel pieces were digested with porcine trypsin (Thermo Fisher Scientific), at a final concentration 12.5 ng/ml in 50 mM ABC, overnight at 37°C. Peptides were extracted using 100% ACN and 0.5% trifluoroacetic acid (Sigma-Aldrich), purified using a Zip Tip (Millipore, Sigma-Aldrich), and dried. Finally, samples were reconstituted in 12 μl of 0.1% formic acid in water (Fisher Chemical) and the peptides were quantified using the Qubit® Protein Assay Kit and the Qubit® 1.0 fluorometer.
Besrour-Aouam N., de Los Rios V., Hernández-Alcántara A.M., Mohedano M.L., Najjari A., López P, & Ouzari H.I. (2023). Proteomic and in silico analyses of dextran synthesis influence on Leuconostoc lactis AV1n adaptation to temperature change. Frontiers in Microbiology, 13, 1077375.
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4

Chemicals for Cultivation of E. coli and Thermococcus

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Chemicals for cultivation of Escherichia coli and Thermococcus sp. strain 2319x1E, such as yeast extract, lysogeny broth (LB), beechwood xylan, D-glucose and D-xylose were obtained from Carl Roth (Germany). Avicel® cellulose, carboxymethyl cellulose (CMC), maltodextrin and soluble starch were purchased from Sigma-Aldrich (United States), xylobiose, ortho-nitrophenyl-β-D-galactopyranoside (ONPG), para-nitrophenyl acetate (PNPA), para-nitrophenyl-β-D-xylopyranoside (PNPX) and para-nitrophenyl- β-D-glucopyranoside (PNPG) from Megazyme (Ireland). Other chemicals which were used in this work are 4’,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, United States), urea (GE Healthcare Life Sciences, Germany), ammonium bicarbonate (ABC; Sigma-Aldrich, United States), dithiothreitol (DTT; Sigma-Aldrich, United States), iodoacetamide (IAM; Sigma-Aldrich, United States), formic acid (FA; Fischer Chemical, Germany) and bovine serum albumin (BSA; VWR Chemicals, United States).
Klaus T., Ninck S., Albersmeier A., Busche T., Wibberg D., Jiang J., Elcheninov A.G., Zayulina K.S., Kaschani F., Bräsen C., Overkleeft H.S., Kalinowski J., Kublanov I.V., Kaiser M, & Siebers B. (2022). Activity-Based Protein Profiling for the Identification of Novel Carbohydrate-Active Enzymes Involved in Xylan Degradation in the Hyperthermophilic Euryarchaeon Thermococcus sp. Strain 2319x1E. Frontiers in Microbiology, 12, 734039.
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5

Quantitative Proteomics Using iTRAQ

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Acetonitrile (ACN), formic acid (FA), water (UHPLC-MS grade), triethylammonium bicarbonate buffer 1 M (TEAB), sodium dodecyl sulfate (SDS), methyl methanethiosulfate (MMTS), trifluoroacetic acid (TFA), ammonium bicarbonate (ABC) and urea were all purchased from Sigma Aldrich (St. Louis, MO, USA). Protease inhibitor cocktail (HALT) and sample cleaning tips (C18) were from Thermo Fisher Scientific (San Jose, CA, USA). BioRad DC protein quantification kit and serum albumin standard were purchased from Bio-Rad (Hercules, CA, USA) and 30 kDa filters from PALL (Port Washington, NY, USA). 8-plex iTRAQ reagent kit and TPCK treated trypsin was acquired from Sciex (SCIEX, Framingham, MA, USA).
Jylhä A., Nättinen J., Aapola U., Mikhailova A., Nykter M., Zhou L., Beuerman R, & Uusitalo H. (2018). Comparison of iTRAQ and SWATH in a clinical study with multiple time points. Clinical Proteomics, 15, 24.
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6

Shotgun Protein Sequencing of Venom

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For shotgun protein sequencing, whole crude venom from a representative individual of each group was reduced, alkylated, digested and submitted for LC-MS/MS analysis. This was performed by first dissolving 10 μg of lyophilized venom in 20 μL of 8 M urea. Samples were then reduced by incubation at 37 °C for 30 min following addition of 10 μL of 15 mM dithiothreitol (DTT) (Sigma-Aldrich, Sydney, Australia), then cysteine residues were alkylated by adding 10 μL of 100 mM iodoacetamide (IAA)(Bio-Rad) and incubating samples in the dark for 30 min at room temperature. 10 μL of 15 mM DTT was then added to quench excess IAA. 50 μL of 50 mM ammonium bicarbonate (ABC)(Sigma-Aldrich, Sydney, Australia) followed by trypsin (Sigma-Aldrich, Sydney, Australia) (1:50) were then added and the sample was incubated overnight at 37 °C to digest venom peptides and proteins. The samples were then desalted using a C18 ZipTip (EMD Millipore, Billerica, MA, USA) according to the manufacturer’s protocol. Dried samples were resuspended in mobile phase A (5% acetonitrile, 0.1% formic acid in deionised water).
Santana R.C., Perez D., Dobson J., Panagides N., Raven R.J., Nouwens A., Jones A., King G.F, & Fry B.G. (2017). Venom Profiling of a Population of the Theraphosid Spider Phlogius crassipes Reveals Continuous Ontogenetic Changes from Juveniles through Adulthood. Toxins, 9(4), 116.
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7

Protein Extraction and Digestion from Organoids

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Organoids were harvested using Cell Recovery Solution (Corning) and multiple ice-cold PBS washes to remove the BME. For protein extraction, a buffer containing 5 M urea (GE Healthcare, Chicago, IL), 50 mM ammonium bicarbonate (ABC) (Sigma-Aldrich) was added to the organoid pellet. Cell lysis was performed by three freeze–thaw cycles, using a −80 °C freezer for freezing and sonication (40 s) in an ultrasonic bath for thawing. Protein concentrations were assessed using Bradford assay, and equal amounts of protein were used for analysis. Protein lysates were reduced with 20 mM Dithiothreitol (DTT) (Sigma-Aldrich) for 45 min, followed by alkylation with 40 mM Iodoacetamide (Sigma-Aldrich) for 45 min in the dark. The alkylation was terminated by 20 mM DDT to consume any excess Iodoacetamide. In solution digestion was performed with a Trypsin/LysC mixture (Promega, Madison, WI), added at a ratio of 1:25 (enzyme:protein), for 2 h at 37 °C in a Thermo Shaker (Grant Instruments, Shepreth, UK) at 250 rpm. The lysate was then diluted to 1 M urea using 50 mM ABC and further digested at 37 °C at 250 rpm overnight. Addition of formic acid (Biosolve, Valkenswaard, The Netherlands) to a total of 1% terminated the digestion.
Kip A.M., Soons Z., Mohren R., Duivenvoorden A.A., Röth A.A., Cillero-Pastor B., Neumann U.P., Dejong C.H., Heeren R.M., Olde Damink S.W, & Lenaerts K. (2021). Proteomics analysis of human intestinal organoids during hypoxia and reoxygenation as a model to study ischemia-reperfusion injury. Cell Death & Disease, 12(1), 95.
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8

Immunoprecipitation of Auxin Receptors

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For immunoprecipitation, ground plant material of pTIR1::TIR1-VENUS in tir1-1 and pAFB1::AFB1-VENUS in afb1-3 transgenic lines was lysed in mild lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 2 mM MgCl2, 0.2 mM EDTA, 1xCPI, 0.5 mM DTT, 0.2% NP40 and 1 mg/ml DNAse) and mildly sonicated using a Bioruptor (Diagenode). After lysate clearance, supernatant was submitted to enrichment using GFP-Trap agarose beads (Chromotek) for 45 minutes at 4°C while gently rotating. Beads were subsequently washed twice in lysis buffer, twice in detergent-free lysis buffer and trice in 50 mM Ammoniumbicarbonate (ABC) (Sigma) with intermediate centrifuging for 2 minutes at 2000g at 4°C. After a final wash, bead-precipitated proteins were alkylated using 50 mM Acrylamide (Sigma). Precipitated proteins were submitted to on-bead trypsin digestion using 0.35 μg trypsin (Roche) per reaction. After overnight incubation at 25°C peptides were desalted and concentrated using C18 Stagetips.
After Stagetip processing, peptides were applied to online nanoLC-MS/MS using a 60 minutes acetonitrile gradient from 8-50%. Spectra were recorded on a LTQ-XL mass spectrometer (Thermo Scientific) according to31
. Statistical analysis using MaxQuant and Perseus software was performed as described previously31
.
Li L., Verstraeten I., Roosjen M., Takahashi K., Rodriguez L., Merrin J., Chen J., Shabala L., Smet W., Ren H., Vanneste S., Shabala S., De Rybel B., Weijers D., Kinoshita T., Gray W.M, & Friml J. (2021). Antagonistic cell surface and intracellular auxin signalling regulate plasma membrane H+-fluxes for root growth. Nature, 599(7884), 273-277.
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9

Glycosphingolipid Analysis Protocol

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Ammonium bicarbonate (ABC), trifluoroacetic acid, cation exchange resin beads (AG50W‐X8), potassium hydroxide, sodium borohydride, and cycloheximide (CHX) were obtained from Sigma‐Aldrich (Steinheim, Germany). The UGCG inhibitor eliglustat was purchased from MedChemExpress (HY‐14885A). The gangliosides GM1a (1545), GM2 (1542), GM3 (1503), GD3 (1504), and GT1b (1548) were purchased from Matreya (State College, PA). SB505124 was purchased from Tocris (3263). HPLC SupraGradient acetonitrile (ACN) was obtained from Biosolve (Valkenswaard, The Netherlands), and other reagents and solvents such as chloroform, methanol, 2‐propanol, and glacial acetic acid were obtained from Merck (Darmstadt, Germany). The 50 mg tC18 reverse phase (RP) cartridges were from Waters (Breda, The Netherlands). Endoglycoceramidase I (EGCase I, recombinant clone derived from Rhodococcus triatomea and expressed in Escherichia coli) and 10× EGCase I buffer (500 mM HEPES, 1 M NaCl, 20 mM DTT, and 0.1% Brij 35, pH 5.2) were purchased from New England BioLabs Inc. (Ipswich, MA). Bafilomycin A1 (BafA1, B1793), cycloheximide (CHX, 01810) and MG132 (474787) were purchased from Sigma‐Aldrich. Biotin (21335) was obtained from Thermo. The concentration of TGF‐β was applied as 2.5 ng/ml, and the same volume of ligand buffer (4 mM HCl, 0.1% BSA) was used as a vehicle control.
Zhang J., van der Zon G., Ma J., Mei H., Cabukusta B., Agaser C.C., Madunić K., Wuhrer M., Zhang T, & ten Dijke P. (2022). ST3GAL5‐catalyzed gangliosides inhibit TGF‐β‐induced epithelial‐mesenchymal transition via TβRI degradation. The EMBO Journal, 42(2), e110553.
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10

Purification and Preparation of Biomolecules

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Deionized water (18.2 MΩ) was purified using a Barnstead Nanopure Infinity system. Tris(2-carboxyethyl)phosphine (TCEP), n-dodecyl-beta-maltoside (DDM) detergent, and protease/phosphatase inhibitor cocktails (catalog 78,430) were purchased from ThermoFisher Scientific. Benzonase nuclease was purchased from EMD Millipore. Magnesium chloride (MgCl2), formic acid (FA), 1× phosphate buffer saline, dimethyl sulfoxide (DMSO), tri-fluoroacetic acid (TFA), ethanol (EtOH), FA, and ammonium bicarbonate (ABC) were purchased from Sigma-Aldrich.
Liao Y.C., Fulcher J.M., Degnan D.J., Williams S.M., Bramer L.M., Veličković D., Zemaitis K.J., Veličković M., Sontag R.L., Moore R.J., Paša-Tolić L., Zhu Y, & Zhou M. (2023). Spatially Resolved Top-Down Proteomics of Tissue Sections Based on a Microfluidic Nanodroplet Sample Preparation Platform. Molecular & Cellular Proteomics : MCP, 22(2), 100491.
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