Serum samples were collected and clotted at room temperature, centrifuged at 1,000 ×g for 3 minutes at 4 °C and stored at −80 °C until further analysis. High-abundance proteins were removed with Pierce Top 12 (cat 85165, Thermo Scientific, Rockform, IL, USA) and the total protein concentration was measured using the bicinchonic acid assay kit (cat 23227, Pierce, Thermo Scientific, Rockform, IL, USA). Total protein (350 µg) was dried in a high-speed vacuum (Savant, Thermofisher) and dissolved in 6 M urea and 100 mM Tris-HCl (pH 7.4). Disulfide bonds were reduced with 10 mM dithiothreitol (cat D9760-1G, DTT, Sigma-Aldrich, St Louis, MO, USA) for 60 minutes at room temperature. The proteins were then alkylated with 30 mM iodoacetamide (cat I6125-5G, Sigma-Aldrich, St Louis, MO, USA) in the dark at room temperature for 60 minutes. The excess iodoacetamide was consumed by adding DTT (30 mM) and the sample was diluted 1:10 with high-purity Milli-Q water (Millipore, Billerica, MA, USA) before trypsin (Promega, Madison, Wisconsin) digestion at 37 °C for 18 hours. The sample was finally purified in Pierce C18 Spin Columns (Thermo Scientific, Rockform, IL, USA), dried, and dissolved in 0.1% formic acid (cat 00940, Sigma-Aldrich, St Louis, MO, USA) containing 12.5 M Hi3 peptide mixture (Waters).
Iodoacetamide
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Sao Tome and Principe, Italy, France, Macao, China, Canada, Australia, Switzerland, Spain
Iodoacetamide is a chemical compound commonly used in biochemistry and molecular biology laboratories. It is a reactive compound that selectively modifies cysteine residues in proteins, thereby allowing for the study of protein structure and function. Iodoacetamide is often used in sample preparation procedures for mass spectrometry and other analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Dithiothreitol (dtt), by Merck Group (502 mentions)
Trypsin, by Promega (255 mentions)
Ammonium bicarbonate, by Merck Group (210 mentions)
Formic acid, by Merck Group (167 mentions)
Urea, by Merck Group (148 mentions)
Trifluoroacetic acid, by Merck Group (105 mentions)
Formic acid, by Thermo Fisher Scientific (98 mentions)
Sequencing grade trypsin, by Promega (81 mentions)
Acetonitrile, by Thermo Fisher Scientific (77 mentions)
Sequencing grade modified trypsin, by Promega (77 mentions)
Acetonitrile, by Merck Group (72 mentions)
Trypsin, by Merck Group (60 mentions)
Dl dithiothreitol, by Merck Group (59 mentions)
Bovine serum albumin (bsa), by Merck Group (43 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (40 mentions)
Sodium chloride (nacl), by Merck Group (32 mentions)
Tris 2 carboxyethyl phosphine, by Merck Group (32 mentions)
Sodium deoxycholate, by Merck Group (31 mentions)
Methanol, by Merck Group (30 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (30 mentions)
1 189 protocols using iodoacetamide
1
Serum Proteome Preparation and Analysis
2
Immunoprecipitation and Ubiquitin Pulldown
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Cells were lysed with radioimmunoprecipitation assay buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.25% sodium deoxycholate, 10 mM iodoacetamide (Sigma-Aldrich), 10 mM N-ethylmaleimide (Sigma-Aldrich), 0.5 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF), 10 μM MG132, and PhosStop phosphatase inhibitors (Sigma-Aldrich). Endogenous Cyclin A2, TFIIB, PRPS1/2/3, CUL5, ACSL4, PCNA, DHPS, and BLVRA were immunoprecipitated with an anti-Cyclin A2 antibody (B-8, sc-271682, SCBT), anti-TFIIB antibody (D-3, sc-271736, SCBT), anti-PRPS1/2/3 antibody (A-11, sc-376440, SCBT), anti-CUL5 antibody (sc-13014, SCBT), anti-ACSL4 antibody (22401-1-AP, Proteintech), anti-PCNA antibody (PC10, sc-56, SCBT), anti-DHPS antibody (A-10, sc-365077, SCBT), and anti-BLVRA antibody (F-1, sc-393385, SCBT) respectively and immunoblotted. Alternatively, His6-ubiquitin was introduced into cells and the cells were lysed with a buffer containing 50 mM Tris-HCl (pH 7.6), 300 mM NaCl, 8 M urea, 0.5% Triton X-100, 10 mM iodoacetamide (Sigma-Aldrich), 10 mM N-ethylmaleimide (Sigma-Aldrich), 0.5 mM AEBSF, 10 μM MG132, and PhosStop phosphatase inhibitors (Sigma-Aldrich). His6-ubiquitin-conjugated proteins were pulled down with Probond resin (Thermo Scientific) and immunoblotted with several antibodies.
3
Trichome Protein Extraction and Digestion
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A volume of 210 μL of trichome-enriched material, 227 μL of trichome-depleted material and 139 μL of whole leaf protein extracts with approximately 150 μg of protein each were digested separately. An appropriate volume of a 100-mM dithiothreitol (Sigma-Aldrich) solution was added to each extract in order to obtain a final concentration of 10 mM of dithiothreitol. Each extract was then vortexed and stored at 45 °C for 45 min. The extracts were subsequently left for 5 minutes at room temperature to cool before adding appropriate volumes of a 90-mM solution of iodoacetamide (Sigma-Aldrich) in order to obtain a final concentration of 30 mM of iodoacetamide. A 50-mM ammonium bicarbonate (Sigma-Aldrich) solution was also added to each extract in order to dilute the concentration of urea to approximately 2 M. All extracts were vortexed and left in the dark for a further 45 min. The pH of each extract was checked using pH strips to confirm that the pH remained within the range of 7.5-8. A sequence-grade trypsin (Promega, Southampton, UK) stock solution (200 ng/μL) was added to each extract to obtain a protein-to-trypsin ratio of 100:1 and all extracts were vortexed and left overnight at 37 °C. The digestion of each extract was stopped by adding 10 μL of 0.1 % trifluoroacetic acid (TFA; Sigma-Aldrich).
4
Synthesis and Characterization of Fullerene Nanomaterials
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Solid C60 and Gd@C82 (99% purity) were purchased from Xiamen Funano Co. Ltd. (Xiamen, China). Anhydrous ethanol, methanol, NaOH, H2O2, HNO3, NaH2PO4, and Na2HPO4 were purchased from Beijing Chemical Works (Beijing, China). NaCl, β-alanine, OA, glutaraldehyde, osmium tetroxide, dithiothreitol, iodoacetamide, pentobarbital sodium, urea, acetonitrile, formic acid, dithiothreitol, iodoacetamide, tetraethylammonium bromide (TEAB), Cyanine5.5 (Cy5.5), and 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nile Red, Hoechst 33342, hematoxylin, eosin, oil red O, and optimal cutting temperature (OCT) compound were purchased from Solarbio (Beijing, China). All reagents and solvents were obtained commercially and used without further purification.
5
Immunoprecipitation and Mass Spectrometry
6
Chromatin Extraction and Protein Purification
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Cells were washed twice with cold PBS buffer supplemented with 5 mM iodoacetamide (Sigma) and 1 mM phenylmethylsulfonylfluoride (Sigma). Cells were scraped directly in 1 ml lysis buffer (50 mM Tris–HCL (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) in the presence of 10 mM N-ethylmaleimide (Sigma), 5 mM iodoacetamide (Sigma), 1 mM phenylmethylsulfonylfluoride (Sigma), 1 mg/mL aprotinin (Sigma), 1 mM leupeptin (Sigma), 1 mM pepstatin (Sigma), transferred into Eppendorf tube and incubated 10 min, 4 °C, with rotation. Lysate was centrifuged at 1200g for 10 min, 4C. Pellet was resuspended in lysis buffer supplemented with 400 mM NaCl (high salt buffer, HSB); chromatin fraction was extracted for 30 min with rotation at 4 °C. The pellet was briefly sonicated using Misonix Sonicator 3000 (2 cycles 10 s on/50 s off, power 2.5) in the experiments with MG132 treatment. Extract was pre-cleared by centrifugation at 16,000g for 10 min, 4 °C and incubated with preconditioned FLAG magnetic beads (Sigma) for 4 h, 4 °C with rotation. Beads were washed six times with HSB and eluted with FLAG peptide.
7
Protein Digestion and Peptide Preparation
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For digestion, 25 μL lysed protein solution (approximately 1 μg/μL) was reduced with 5 mM dithiothreitol (by adding 0.125 μL 1 M dithiothreitol (Sigma) for 30 min at 56 °C. Then the protein solution was alkylated with 11 mM Iodoacetamide (by adding 0.5 μL 0.55 M Iodoacetamide (Sigma)) for 15 min at room temperature in darkness. The protein sample was diluted by adding 100 μL 100 mM triethylammonium bicarbonate (TEAB) to a urea concentration less than 2 M. Finally, trypsin [(Promega)] was added at a 1:50 trypsin-to-protein mass ratio for the first digestion overnight and a 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion. After the second 4 h enzymatic hydrolysis, the reaction was terminated by adding 10% trifluoroacetic acid (TFA) solution and adjusting the pH to 2–3. Then, the peptides were desalted by the C18 SPE column. Desalted peptides were frozen, drained off, and dissolved by mass spectrometry mobile phase A. Finally, 1 μg of peptides were taken to put on the machine.
8
Protein Preparation for Mass Spectrometry
9
Preparation of Alkylated Protein Digests
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Pooled gel filtration fractions of
extracted cross-linked proteins in the 400 kDa to 1 to 2 MDa range
were concentrated to ∼10 mg protein/mL with 0.5 mL of Amicon
Ultra 10 kDa cut off centrifugal filters (Millipore). Prior to digestion,
cysteines were alkylated by the addition of a solution of 0.8 M iodoacetamide
(Sigma–Aldrich), followed by the addition of solutions of 1
M Tris-HCl pH 8.0 and 9.6 M urea (Bioreagent grade, Sigma–Aldrich)
to obtain final concentrations of 40 mM iodoacetamide, 0.1 M Tris
HCl and 6 M urea, respectively. Incubation was for 30 min at room
temperature in the dark. The solution was diluted six times by the
addition of 0.1 M Tris–HCl pH 8.0 and digested with trypsin
(Trypsin Gold, Promega, Madison, WI) overnight at 30 °C at a
1:50 (w/w) ratio of enzyme and substrate. Peptides were desalted on
C18 reversed-phase TT3 top tips (Glygen, Columbia, MD), eluted with
0.1% TFA in 50% acetonitrile, and dried in a vacuum centrifuge.
extracted cross-linked proteins in the 400 kDa to 1 to 2 MDa range
were concentrated to ∼10 mg protein/mL with 0.5 mL of Amicon
Ultra 10 kDa cut off centrifugal filters (Millipore). Prior to digestion,
cysteines were alkylated by the addition of a solution of 0.8 M iodoacetamide
(Sigma–Aldrich), followed by the addition of solutions of 1
M Tris-HCl pH 8.0 and 9.6 M urea (Bioreagent grade, Sigma–Aldrich)
to obtain final concentrations of 40 mM iodoacetamide, 0.1 M Tris
HCl and 6 M urea, respectively. Incubation was for 30 min at room
temperature in the dark. The solution was diluted six times by the
addition of 0.1 M Tris–HCl pH 8.0 and digested with trypsin
(Trypsin Gold, Promega, Madison, WI) overnight at 30 °C at a
1:50 (w/w) ratio of enzyme and substrate. Peptides were desalted on
C18 reversed-phase TT3 top tips (Glygen, Columbia, MD), eluted with
0.1% TFA in 50% acetonitrile, and dried in a vacuum centrifuge.
10
Protein Gel Electrophoresis and Mass Spectrometry
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All reagents used were HPLC grade or electrophoresis grade. Acrylamide/bis-acrylamide 30% solution (37.5:1), ammonium bicarbonate (ambic), β-mercaptoethanol, Coomassie Brilliant Blue R250 (CBB), dithiothreitol (DTT), iodoacetamide (IAA), formic acid, glycerol 86–88%, sodium borohydride (NaBH4), sodium carbonate, sodium citrate tribasic dihydrate (HOC(COONa)(CH2COONa)2·2H2O), (N,N,N, N′-tetramethylethylenediamine (TMED), trifluoroacetic acid, tris-base, trypsin, and the Sigma Marker wide range 6.5–200 KDa were all from Merck (Barcelona, Spain). Acetonitrile, formaldehyde, methanol and sodium dodecyl sulfate (SDS) were supplied by Panreac Química SLU (Barcelona, Spain). Bromophenol-blue was purchased from Riedel-de Haen (Seelze, Germany). Pierce™ trypsin Protease, MS Grade was purchased from Thermo Fisher Scientific (Bremen, Spain). Hydrogen tetrachloroaurate (III) hydrate (HAuCl4·xH2O) (99.9% Au) (49% Au) at 10% w/v was acquired from Strem Chemicals (Kehl, Germany).
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