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GE Healthcare iodoacetamide
Iodoacetamide, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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iodoacetamide - by Bioz Stars, 2020-02
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Centrifugation:

Article Title: Quantitative Proteome Analysis of Human Plasma Following in vivo Lipopolysaccharide Administration using 16O/18O Labeling and the Accurate Mass and Time Tag Approach
Article Snippet: The plasma samples were prepared from whole blood by centrifugation; samples at T = 0 h (control, baseline immediately prior to endotoxin administration) and T = 9 h (LPS-treated, 9 h following LPS administration) were used for this study. .. Aliquots of 200 μL each of the control and LPS-treated plasma samples were diluted and denatured using 8 M urea, 50 mM NH4 HCO3 , pH 8.2 for 1 h at 37° C and reduced with 10 mM dithiothreitol (DTT) for 30 min at 37° C. Protein cysteinyl residues were alkylated with 40 mM iodoacetamide for 90 min at room temperature, and samples were desalted using a pre-packed PD-10 column containing Sephadex G-25 (Amersham Biosciences, Piscataway, NJ).

Article Title: The Schistosomiasis SpleenOME: Unveiling the Proteomic Landscape of Splenomegaly Using Label-Free Mass Spectrometry
Article Snippet: Alkylation was achieved using iodoacetamide (GE Healthcare, Little Chalfont, Buckinghamshire, UK) to a final concentration of 9.4 mM at room temperature in the dark, for 30min. .. After that, a centrifugation step was performed at 20,000 g for 15min at 7°C (Mikro 200R, Hettich Zentrifugen, Tuttlingen, DEU) for the removal of insoluble particles.

Nucleic Acid Electrophoresis:

Article Title: Proteomic analysis reveals virus-specific Hsp25 modulation in cardiac myocytes
Article Snippet: Paragraph title: Reagents for 2-dimensional difference gel electrophoresis (2D-DIGE) ... Urea, thiourea, SDS, DTT, Tris, and iodoacetamide were purchased from GE Healthcare.

Article Title: Involvement of oxidative modification of proteins related to ATP synthesis in the left ventricles of hamsters with cardiomyopathy
Article Snippet: .. After reduction and alkylation of disulfide bonds with 10 mg/ml DTT and 25 mg/ml iodoacetamide, respectively, the second-dimension 12.5% SDS–polyacrylamide gel electrophoresis was run on an Ettan DALT Six large electrophoresis system (GE Healthcare). .. After the second-dimension, the proteins from the gels were transferred to polyvinylidene fluoride (PVDF) membrane (Immobilon-P transfer membrane; Millipore) using a TE77 semidry transfer unit (GE Healthcare) at 50 V for 30 min.

Electrophoresis:

Article Title: Involvement of oxidative modification of proteins related to ATP synthesis in the left ventricles of hamsters with cardiomyopathy
Article Snippet: .. After reduction and alkylation of disulfide bonds with 10 mg/ml DTT and 25 mg/ml iodoacetamide, respectively, the second-dimension 12.5% SDS–polyacrylamide gel electrophoresis was run on an Ettan DALT Six large electrophoresis system (GE Healthcare). .. After the second-dimension, the proteins from the gels were transferred to polyvinylidene fluoride (PVDF) membrane (Immobilon-P transfer membrane; Millipore) using a TE77 semidry transfer unit (GE Healthcare) at 50 V for 30 min.

Incubation:

Article Title: A-Kinase Anchoring Protein 79/150 Scaffolds Transient Receptor Potential A 1 Phosphorylation and Sensitization by Metabotropic Glutamate Receptor Activation
Article Snippet: In-gel digestion of proteins The excised protein gel band was cut to small particles with a spot picker and washed twice with HPLC-grade water (Fisher) and incubated with 1:1 v/v of 0.1 NH4 HCO3 (Sigma) for 15 min. Wash solution was removed and HPLC-grade acetonitrile (Fisher) was then added to cover the gel particles for 1 hr, acetonitrile removed and gel particles were rehydrated in 0.1 M NH4 HCO3 for 10 min. An equal volume of ACN was then added for a 1:1 v/v of 0.1 NH4 HCO3 /ACN. .. After 10 min all liquid was removed and the gel particles dried under vacuum then reduced with 10 mM dithiothreitol (DTT, Thermo Scientific) and alkylated with 55 mM iodoacetamide (GE Healthcare) in 0.1 M NH4HCO3.

Article Title: HnRNPK/miR-223/FBXW7 feedback cascade promotes pancreatic cancer cell growth and invasion
Article Snippet: The cells were treated with MG132 for 6 h, harvested, and lysed with RIPA buffer supplemented with 10 mM iodoacetamide (GE Healthcare) and protease inhibitors. .. 2 μL in vitro translated hnRNPK was incubated in a 30 μL reaction mixture containing 5 mM MgCl2, 0.6 mM DTT, 2 mM ATP, 50 mM Tris·HCl (pH 7.5), 100ng of ubiquitin-activating enzyme E1, 200 ng of ubiquitin-conjugating enzyme UbcH5c, 10 μg of ubiquitin (Calbiochem), and ~2 μg of FBXW7.

Article Title: Proteomic Analysis of Paracoccidioides brasiliensis During Infection of Alveolar Macrophages Primed or Not by Interferon-Gamma
Article Snippet: Next, 75 μL of RapiGESTTM SF Surfactante (0.2% v/v) (Waters Corporation, Billerica, MA, United States) was added and the sample was vortexed and incubated in a dry bath at 80°C for 15 min. .. In each sample were added 2.5 μL of 100 mM dithiothreitol (GE Healthcare, Piscataway, NJ, United States), at 60°C for 30 min, while cysteines were alkylated by the addition of 2.5 μL of 300 mM iodoacetamide (GE Healthcare, Piscataway, NJ, United States) for 30 min, at room temperature in the dark.

Article Title: Nanobubbles as ultrasound contrast agent for facilitating small cell lung cancer imaging
Article Snippet: Identification of nanobubbles Briefly, the dried solutions were incubated with 50 mM Tris [2-carboxyethyl]phosphine (TCEP, Sigma) in 25 mM NH4 HCO3 at 56°Cfor 1 hour to reductively cleave the disulfide bonds of proteins. .. The resulting sulfhydryl functional groups were alkylated with 100 mM iodoacetamide (IAA, Amersham) in 25 mM NH4 HCO3 at room temperature for 0.5 hour in the dark.

Article Title: The Schistosomiasis SpleenOME: Unveiling the Proteomic Landscape of Splenomegaly Using Label-Free Mass Spectrometry
Article Snippet: Briefly, samples were added with RapiGest to a final concentration of (0.06% w/v) and incubated at 80°C for 10min. .. Alkylation was achieved using iodoacetamide (GE Healthcare, Little Chalfont, Buckinghamshire, UK) to a final concentration of 9.4 mM at room temperature in the dark, for 30min.

Article Title: Comparative Proteomics Analysis of Two Strains of Neisseria meningitidis Serogroup B and Neisseria lactamica
Article Snippet: .. This step was followed by a 15-minute incubation in equilibration buffer plus 2.5% (w/v) iodoacetamide (GE Healthcare, USA). .. The second dimension was run on a 12% acrylamide gradient gel (18 × 20 cm × 1.5 mm) with a constant current of 20 mA, using a Mini-PROTEAN 3 Cell (Bio-Rad, USA), until the dye reached the bottom of the gel.

Electric Cell-substrate Impedance Sensing:

Article Title: Assessment of electrophile damage in a human brain endothelial cell line utilizing a clickable alkyne analogue of 2-chlorohexadecanal
Article Snippet: Electrical cell-substrate impedance sensing (ECIS) electrode arrays (8W10E+) were from Ibidi (Martinsried, Germany). .. Urea, thiourea, iodoacetamide, immobilized pH gradient strips (IPG strips, pH 3–10), and Pharmalyte (pH 3–10) were from GE Healthcare (Amersham Biosciences, Vienna).

Activity Assay:

Article Title: Quantitative Proteome Analysis of Human Plasma Following in vivo Lipopolysaccharide Administration using 16O/18O Labeling and the Accurate Mass and Time Tag Approach
Article Snippet: Aliquots of 200 μL each of the control and LPS-treated plasma samples were diluted and denatured using 8 M urea, 50 mM NH4 HCO3 , pH 8.2 for 1 h at 37° C and reduced with 10 mM dithiothreitol (DTT) for 30 min at 37° C. Protein cysteinyl residues were alkylated with 40 mM iodoacetamide for 90 min at room temperature, and samples were desalted using a pre-packed PD-10 column containing Sephadex G-25 (Amersham Biosciences, Piscataway, NJ). .. Tryptic activity of residual trypsin was quenched by boiling the samples for 10 min and immediately placing the samples on ice.

Mass Spectrometry:

Article Title: Comprehensive Characterization of Glycosylation and Hydroxylation of Basement Membrane Collagen IV by High-Resolution Mass Spectrometry
Article Snippet: Ammonium bicarbonate, sodium phosphate, and MS grade formic acid and dithiothreitol (DTT) were purchased from Sigma-Aldrich (St. Louis, MO). .. Iodoacetamide was acquired from GE Healthcare (Piscataway, NJ).

Article Title: Proteomic Analysis of Paracoccidioides brasiliensis During Infection of Alveolar Macrophages Primed or Not by Interferon-Gamma
Article Snippet: In each sample were added 2.5 μL of 100 mM dithiothreitol (GE Healthcare, Piscataway, NJ, United States), at 60°C for 30 min, while cysteines were alkylated by the addition of 2.5 μL of 300 mM iodoacetamide (GE Healthcare, Piscataway, NJ, United States) for 30 min, at room temperature in the dark. .. The tryptic peptides were analyzed using a nanoACQUITY UPLC® M-Class system (Waters Corporation, Billerica, MA, United States) coupled to Synapt G1 MSTM mass spectrometer (Waters Corporation, Billerica, MA, United States), equipped with a NanoElectronSpray source and two mass analyzers: a quadrupole and a time-of-flight (TOF) operating in the V-mode.

Article Title: Nanobubbles as ultrasound contrast agent for facilitating small cell lung cancer imaging
Article Snippet: The resulting sulfhydryl functional groups were alkylated with 100 mM iodoacetamide (IAA, Amersham) in 25 mM NH4 HCO3 at room temperature for 0.5 hour in the dark. .. The pooled extracts were evaporated in a vacuum centrifuge (Labconco, Kansas, MO), and resuspended in 0.1% methanoic acid (Sigma) prior to the LC separation and MS detection.

Article Title: The Schistosomiasis SpleenOME: Unveiling the Proteomic Landscape of Splenomegaly Using Label-Free Mass Spectrometry
Article Snippet: Protein extracts were reduced and alkylated before digestion with MS grade trypsin (Promega, Madison, WI, USA) in the presence of the digestion enhancer RapiGest (Waters, UK). .. Alkylation was achieved using iodoacetamide (GE Healthcare, Little Chalfont, Buckinghamshire, UK) to a final concentration of 9.4 mM at room temperature in the dark, for 30min.

BIA-KA:

Article Title: Quantitative Proteome Analysis of Human Plasma Following in vivo Lipopolysaccharide Administration using 16O/18O Labeling and the Accurate Mass and Time Tag Approach
Article Snippet: Aliquots of 200 μL each of the control and LPS-treated plasma samples were diluted and denatured using 8 M urea, 50 mM NH4 HCO3 , pH 8.2 for 1 h at 37° C and reduced with 10 mM dithiothreitol (DTT) for 30 min at 37° C. Protein cysteinyl residues were alkylated with 40 mM iodoacetamide for 90 min at room temperature, and samples were desalted using a pre-packed PD-10 column containing Sephadex G-25 (Amersham Biosciences, Piscataway, NJ). .. The protein concentrations for the desalted samples were measured using a BCA protein assay (Pierce, Rockford, IL) that gave total protein amounts of 15.0 mg and 13.9 mg for the control and LPS-treated plasma samples, respectively.

Modification:

Article Title: Quantitative Proteomics of Breast Tumors: Tissue Quality Assessment to Clinical Biomarkers
Article Snippet: The human MCF7 (ER+ ), T47D (PR+ ), BT474 (HER2+ ), and HCC38 (triple negative) breast cancer cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA) for assay development against the proteins listed in parentheses and grown under the recommended conditions: 37°C, 5% CO2 , Dulbecco’s modified Eagle’s (D5796, Sigma, St. Louis, MO) or RPMI-1640 culture media (R8758, Sigma, St. Louis, MO), supplemented with 10% fetal bovine serum (SH30910.03, Thermo Scientific) and 1% penicillin/streptomycin (P0781 Sigma, St. Louis, MO). β-Estradiol (E2758), NaF (S7920), Na2 MoO4 (243655), acetic acid (A6283), Coomassie Brilliant Blue G (B5133), ammonium bicarbonate (A6141), and tris(2-carboxyethyl)phosphine hydrochloride (C4706) were purchased from Sigma-Aldrich (St. Louis, MO). .. Iodoacetamide (RPN6302V) was purchased from GE Healthcare (Chicago, IL).

Article Title: Nanobubbles as ultrasound contrast agent for facilitating small cell lung cancer imaging
Article Snippet: The resulting sulfhydryl functional groups were alkylated with 100 mM iodoacetamide (IAA, Amersham) in 25 mM NH4 HCO3 at room temperature for 0.5 hour in the dark. .. Subsequently, the proteins were digested with 20 ng/μl porcine trypsin (modified proteomics grade, Sigma) at 37°C overnight.

Article Title: Involvement of oxidative modification of proteins related to ATP synthesis in the left ventricles of hamsters with cardiomyopathy
Article Snippet: Paragraph title: Detection of carbonyl modification of proteins (2D-oxyblot analysis) ... After reduction and alkylation of disulfide bonds with 10 mg/ml DTT and 25 mg/ml iodoacetamide, respectively, the second-dimension 12.5% SDS–polyacrylamide gel electrophoresis was run on an Ettan DALT Six large electrophoresis system (GE Healthcare).

High Performance Liquid Chromatography:

Article Title: A-Kinase Anchoring Protein 79/150 Scaffolds Transient Receptor Potential A 1 Phosphorylation and Sensitization by Metabotropic Glutamate Receptor Activation
Article Snippet: In-gel digestion of proteins The excised protein gel band was cut to small particles with a spot picker and washed twice with HPLC-grade water (Fisher) and incubated with 1:1 v/v of 0.1 NH4 HCO3 (Sigma) for 15 min. Wash solution was removed and HPLC-grade acetonitrile (Fisher) was then added to cover the gel particles for 1 hr, acetonitrile removed and gel particles were rehydrated in 0.1 M NH4 HCO3 for 10 min. An equal volume of ACN was then added for a 1:1 v/v of 0.1 NH4 HCO3 /ACN. .. After 10 min all liquid was removed and the gel particles dried under vacuum then reduced with 10 mM dithiothreitol (DTT, Thermo Scientific) and alkylated with 55 mM iodoacetamide (GE Healthcare) in 0.1 M NH4HCO3.

Article Title: Proteomic analysis reveals virus-specific Hsp25 modulation in cardiac myocytes
Article Snippet: Urea, thiourea, SDS, DTT, Tris, and iodoacetamide were purchased from GE Healthcare. .. Methanol, acetonitrile, and HPLC grade water were obtained from Burdick & Jackson.

Article Title: Comprehensive Characterization of Glycosylation and Hydroxylation of Basement Membrane Collagen IV by High-Resolution Mass Spectrometry
Article Snippet: Iodoacetamide was acquired from GE Healthcare (Piscataway, NJ). .. HPLC-grade solvents, including water, methanol, and acetonitrile, were procured from Sigma-Aldrich (St. Louis, MO).

Immunoprecipitation:

Article Title: HnRNPK/miR-223/FBXW7 feedback cascade promotes pancreatic cancer cell growth and invasion
Article Snippet: The cells were treated with MG132 for 6 h, harvested, and lysed with RIPA buffer supplemented with 10 mM iodoacetamide (GE Healthcare) and protease inhibitors. .. Endogenous hnRNPK was immunoprecipitated with 1 μg of anti-hnRNPK polyclonal antibody and immunoblotted with anti-HA or indicated antibodies.

Protease Inhibitor:

Article Title: Proteomic analysis reveals virus-specific Hsp25 modulation in cardiac myocytes
Article Snippet: Urea, thiourea, SDS, DTT, Tris, and iodoacetamide were purchased from GE Healthcare. .. Complete Mini Protease Inhibitor Cocktail Tablets were obtained from Roche Applied Science.

Sequencing:

Article Title: Quantitative Proteome Analysis of Human Plasma Following in vivo Lipopolysaccharide Administration using 16O/18O Labeling and the Accurate Mass and Time Tag Approach
Article Snippet: Aliquots of 200 μL each of the control and LPS-treated plasma samples were diluted and denatured using 8 M urea, 50 mM NH4 HCO3 , pH 8.2 for 1 h at 37° C and reduced with 10 mM dithiothreitol (DTT) for 30 min at 37° C. Protein cysteinyl residues were alkylated with 40 mM iodoacetamide for 90 min at room temperature, and samples were desalted using a pre-packed PD-10 column containing Sephadex G-25 (Amersham Biosciences, Piscataway, NJ). .. The samples were then digested into peptides using sequencing grade trypsin (Promega, Madison, WI) overnight at 37° C with a 1:50 (w:w) trypsin-to-protein ratio.

Article Title: Assessment of electrophile damage in a human brain endothelial cell line utilizing a clickable alkyne analogue of 2-chlorohexadecanal
Article Snippet: Urea, thiourea, iodoacetamide, immobilized pH gradient strips (IPG strips, pH 3–10), and Pharmalyte (pH 3–10) were from GE Healthcare (Amersham Biosciences, Vienna). .. Sequencing grade Trypsin was from Promega (Mannheim, Germany).

Article Title: The Schistosomiasis SpleenOME: Unveiling the Proteomic Landscape of Splenomegaly Using Label-Free Mass Spectrometry
Article Snippet: Alkylation was achieved using iodoacetamide (GE Healthcare, Little Chalfont, Buckinghamshire, UK) to a final concentration of 9.4 mM at room temperature in the dark, for 30min. .. Samples were digested using sequencing grade trypsin at a ratio of 50:1 (protein/trypsin) and incubated at 37°C for 16 h. To stop the digestion and precipitate RapiGest, trifluoroacetic acid was added to a final concentration of 0.5% v/v, resulting in a pH ≤ 2.

MTT Assay:

Article Title: Assessment of electrophile damage in a human brain endothelial cell line utilizing a clickable alkyne analogue of 2-chlorohexadecanal
Article Snippet: Dimethylsulfoxide (DMSO), 1,3-diaminopropane, dichloromethane, trimethylamine, oxalyl chloride, N-chlorosuccinimide, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), pentafluorobenzyl (PFB) hydroxylamine, PFB bromide, pentafluorobenzoyl (PFBoyl), sodium cyanoborohydride, dithiothreitol (DTT), and DL-proline, were from Sigma-Aldrich (Vienna). .. Urea, thiourea, iodoacetamide, immobilized pH gradient strips (IPG strips, pH 3–10), and Pharmalyte (pH 3–10) were from GE Healthcare (Amersham Biosciences, Vienna).

Labeling:

Article Title: The liposoluble proteome of Mycoplasma agalactiae: an insight into the minimal protein complement of a bacterial membrane
Article Snippet: Labeled samples were mixed, IPG buffer corresponding to the desired pH range was added at a 1% final concentration, and DeStreak Rehydration Solution (GE Healthcare) was added to a total volume of 340 μL. .. After focusing, strips were equilibrated in 50 mM Tris-HCl, pH 6.8, 2% SDS, 7 M urea, 10% glycerol, supplemented with 2% DTT for 10 min, and then with 2.5% iodoacetamide for 10 min. Proteins were then subjected to SDS-PAGE in 10-18% gradient polyacrylamide gels on the Ettan DALTsix system (GE Healthcare), following the manufacturer instructions.

Article Title: Reduced Tau protein expression is associated with frontotemporal degeneration with progranulin mutation
Article Snippet: Finally, the internal standard labeled with Cy2 and the samples labeled with either Cy3 or Cy5 were pooled and the final volume was adjusted to 350 μL by the addition of rehydration buffer [Urea 8 M, Thiourea 2 M, CHAPS 2 %, Destreak reagent 1.1 % (GE Lifesciences), IPG buffer pH 3–11 1.2 % (GE Lifesciences), bromophenol blue 0.01 %]. .. Strips were then equilibrated in equilibration buffer (Urea 6 M, SDS 2 %, Glycerol 30 %, Tris–HCl 50 mM, pH 8.6) with successively 1 % DTT (dithiothreitol) and 4.7 % iodoacetamide for 15 min. Proteins were then separated in the second dimension on 1 mm-thick 12 % SDS-PAGE gels in an ETTAN DALTSix system (GE Lifesciences).

Article Title: Involvement of oxidative modification of proteins related to ATP synthesis in the left ventricles of hamsters with cardiomyopathy
Article Snippet: Carbonylated proteins were labeled by derivatization of carbonyl group with 2,4-denitrophenylhydrazone (DNP) by reaction with DNPH. .. After reduction and alkylation of disulfide bonds with 10 mg/ml DTT and 25 mg/ml iodoacetamide, respectively, the second-dimension 12.5% SDS–polyacrylamide gel electrophoresis was run on an Ettan DALT Six large electrophoresis system (GE Healthcare).

Purification:

Article Title: HnRNPK/miR-223/FBXW7 feedback cascade promotes pancreatic cancer cell growth and invasion
Article Snippet: The cells were treated with MG132 for 6 h, harvested, and lysed with RIPA buffer supplemented with 10 mM iodoacetamide (GE Healthcare) and protease inhibitors. .. Or a 100 μL sample of the in vitro translated FLAG-tagged FBXW7 was purified using Flag beads, and the reconstituted complex was used for in vitro ubiquitination without elution from the Flag beads.

Staining:

Article Title: Comparative Proteomics Analysis of Two Strains of Neisseria meningitidis Serogroup B and Neisseria lactamica
Article Snippet: This step was followed by a 15-minute incubation in equilibration buffer plus 2.5% (w/v) iodoacetamide (GE Healthcare, USA). .. The gels were stained with colloidal Coomassie brilliant blue G-250 (Bio-Rad, USA), as previously described ( ).

SDS Page:

Article Title: The liposoluble proteome of Mycoplasma agalactiae: an insight into the minimal protein complement of a bacterial membrane
Article Snippet: .. After focusing, strips were equilibrated in 50 mM Tris-HCl, pH 6.8, 2% SDS, 7 M urea, 10% glycerol, supplemented with 2% DTT for 10 min, and then with 2.5% iodoacetamide for 10 min. Proteins were then subjected to SDS-PAGE in 10-18% gradient polyacrylamide gels on the Ettan DALTsix system (GE Healthcare), following the manufacturer instructions. .. DIGE images were detected with a Typhoon Scanner (GE Healthcare) and processed with DeCyder (GE Healthcare) for image analysis.

Article Title: Reduced Tau protein expression is associated with frontotemporal degeneration with progranulin mutation
Article Snippet: .. Strips were then equilibrated in equilibration buffer (Urea 6 M, SDS 2 %, Glycerol 30 %, Tris–HCl 50 mM, pH 8.6) with successively 1 % DTT (dithiothreitol) and 4.7 % iodoacetamide for 15 min. Proteins were then separated in the second dimension on 1 mm-thick 12 % SDS-PAGE gels in an ETTAN DALTSix system (GE Lifesciences). .. Fluorescently labeled protein spots were visualized using a Typhon FLA 9500 imager (GE Lifesciences).

Article Title: Proteomic Profiling for Peritoneal Dialysate: Differential Protein Expression in Diabetes Mellitus
Article Snippet: Then the strips were subjected to a two-step equilibration in buffers: the first step containing 6 M urea, 30% glycerol, 2% SDS, and 50 mM Tris-HCl (pH 8.8) with 1% w/v dithiothreitol (DTT, Affymetrix/USB, 15395) and for the second step, with 2.5% w/v iodoacetamide (IAA, Amersham Biosciences, RPN6302V). .. The strips were then transferred onto the second-dimensional SDS-PAGE equipment and developed on 1.0 mm thick gradient (8–16%) polyacrylamide gels at 10°C.

Software:

Article Title: Reduced Tau protein expression is associated with frontotemporal degeneration with progranulin mutation
Article Snippet: Strips were then equilibrated in equilibration buffer (Urea 6 M, SDS 2 %, Glycerol 30 %, Tris–HCl 50 mM, pH 8.6) with successively 1 % DTT (dithiothreitol) and 4.7 % iodoacetamide for 15 min. Proteins were then separated in the second dimension on 1 mm-thick 12 % SDS-PAGE gels in an ETTAN DALTSix system (GE Lifesciences). .. Gels were scanned at 200 μm resolution and images were exported for further analysis using SameSpots (TotalLab) software.

Article Title: Involvement of oxidative modification of proteins related to ATP synthesis in the left ventricles of hamsters with cardiomyopathy
Article Snippet: After reduction and alkylation of disulfide bonds with 10 mg/ml DTT and 25 mg/ml iodoacetamide, respectively, the second-dimension 12.5% SDS–polyacrylamide gel electrophoresis was run on an Ettan DALT Six large electrophoresis system (GE Healthcare). .. Then, the spot intensities of carbonyl proteins were quantified using PDQuest version 8.0 Advanced 2-D Analysis software (Bio-Rad Laboratories).

Ubiquitin Assay:

Article Title: HnRNPK/miR-223/FBXW7 feedback cascade promotes pancreatic cancer cell growth and invasion
Article Snippet: Paragraph title: Ubiquitination assay ... The cells were treated with MG132 for 6 h, harvested, and lysed with RIPA buffer supplemented with 10 mM iodoacetamide (GE Healthcare) and protease inhibitors.

Functional Assay:

Article Title: Nanobubbles as ultrasound contrast agent for facilitating small cell lung cancer imaging
Article Snippet: .. The resulting sulfhydryl functional groups were alkylated with 100 mM iodoacetamide (IAA, Amersham) in 25 mM NH4 HCO3 at room temperature for 0.5 hour in the dark. .. Subsequently, the proteins were digested with 20 ng/μl porcine trypsin (modified proteomics grade, Sigma) at 37°C overnight.

Sample Prep:

Article Title: Quantitative Proteome Analysis of Human Plasma Following in vivo Lipopolysaccharide Administration using 16O/18O Labeling and the Accurate Mass and Time Tag Approach
Article Snippet: Paragraph title: Human Plasma Sample Preparation ... Aliquots of 200 μL each of the control and LPS-treated plasma samples were diluted and denatured using 8 M urea, 50 mM NH4 HCO3 , pH 8.2 for 1 h at 37° C and reduced with 10 mM dithiothreitol (DTT) for 30 min at 37° C. Protein cysteinyl residues were alkylated with 40 mM iodoacetamide for 90 min at room temperature, and samples were desalted using a pre-packed PD-10 column containing Sephadex G-25 (Amersham Biosciences, Piscataway, NJ).

In Vitro:

Article Title: HnRNPK/miR-223/FBXW7 feedback cascade promotes pancreatic cancer cell growth and invasion
Article Snippet: The cells were treated with MG132 for 6 h, harvested, and lysed with RIPA buffer supplemented with 10 mM iodoacetamide (GE Healthcare) and protease inhibitors. .. Or a 100 μL sample of the in vitro translated FLAG-tagged FBXW7 was purified using Flag beads, and the reconstituted complex was used for in vitro ubiquitination without elution from the Flag beads.

Concentration Assay:

Article Title: The Schistosomiasis SpleenOME: Unveiling the Proteomic Landscape of Splenomegaly Using Label-Free Mass Spectrometry
Article Snippet: .. Alkylation was achieved using iodoacetamide (GE Healthcare, Little Chalfont, Buckinghamshire, UK) to a final concentration of 9.4 mM at room temperature in the dark, for 30min. .. Samples were digested using sequencing grade trypsin at a ratio of 50:1 (protein/trypsin) and incubated at 37°C for 16 h. To stop the digestion and precipitate RapiGest, trifluoroacetic acid was added to a final concentration of 0.5% v/v, resulting in a pH ≤ 2.

Article Title: The liposoluble proteome of Mycoplasma agalactiae: an insight into the minimal protein complement of a bacterial membrane
Article Snippet: Labeled samples were mixed, IPG buffer corresponding to the desired pH range was added at a 1% final concentration, and DeStreak Rehydration Solution (GE Healthcare) was added to a total volume of 340 μL. .. After focusing, strips were equilibrated in 50 mM Tris-HCl, pH 6.8, 2% SDS, 7 M urea, 10% glycerol, supplemented with 2% DTT for 10 min, and then with 2.5% iodoacetamide for 10 min. Proteins were then subjected to SDS-PAGE in 10-18% gradient polyacrylamide gels on the Ettan DALTsix system (GE Healthcare), following the manufacturer instructions.

Two-Dimensional Gel Electrophoresis:

Article Title: Comparative Proteomics Analysis of Two Strains of Neisseria meningitidis Serogroup B and Neisseria lactamica
Article Snippet: Paragraph title: 3.3. Two-Dimensional Gel Electrophoresis (2-DE) ... This step was followed by a 15-minute incubation in equilibration buffer plus 2.5% (w/v) iodoacetamide (GE Healthcare, USA).

Article Title: Proteomic Profiling for Peritoneal Dialysate: Differential Protein Expression in Diabetes Mellitus
Article Snippet: Paragraph title: 2.2. Two-Dimensional Gel Electrophoresis (2DE) ... Then the strips were subjected to a two-step equilibration in buffers: the first step containing 6 M urea, 30% glycerol, 2% SDS, and 50 mM Tris-HCl (pH 8.8) with 1% w/v dithiothreitol (DTT, Affymetrix/USB, 15395) and for the second step, with 2.5% w/v iodoacetamide (IAA, Amersham Biosciences, RPN6302V).

Lysis:

Article Title: Involvement of oxidative modification of proteins related to ATP synthesis in the left ventricles of hamsters with cardiomyopathy
Article Snippet: To reduce artefactual oxidation of the samples, we added thiourea in lysis buffer. .. After reduction and alkylation of disulfide bonds with 10 mg/ml DTT and 25 mg/ml iodoacetamide, respectively, the second-dimension 12.5% SDS–polyacrylamide gel electrophoresis was run on an Ettan DALT Six large electrophoresis system (GE Healthcare).

Electrofocusing:

Article Title: Comparative Proteomics Analysis of Two Strains of Neisseria meningitidis Serogroup B and Neisseria lactamica
Article Snippet: Two-Dimensional Gel Electrophoresis (2-DE) First dimension Isoelectric Focusing (IEF) was performed using the PROTEAN IEF Cell (Bio-Rad, USA). .. This step was followed by a 15-minute incubation in equilibration buffer plus 2.5% (w/v) iodoacetamide (GE Healthcare, USA).

Article Title: The liposoluble proteome of Mycoplasma agalactiae: an insight into the minimal protein complement of a bacterial membrane
Article Snippet: IEF was carried out on an Ettan IPGphor II (GE Healthcare) for a total of ~60,000 Vh. .. After focusing, strips were equilibrated in 50 mM Tris-HCl, pH 6.8, 2% SDS, 7 M urea, 10% glycerol, supplemented with 2% DTT for 10 min, and then with 2.5% iodoacetamide for 10 min. Proteins were then subjected to SDS-PAGE in 10-18% gradient polyacrylamide gels on the Ettan DALTsix system (GE Healthcare), following the manufacturer instructions.

Article Title: Proteomic Profiling for Peritoneal Dialysate: Differential Protein Expression in Diabetes Mellitus
Article Snippet: IEF strips (pH 3–10, IPGphor, Amersham Biosciences, Uppsala, Sweden) were developed through a stepwise incremental voltage program: 30 V for 16 hr, 500 V for 1 hr, 1000 V for 1 hr, and 8000 V for 4 hr, with a total power of 34 kV hr. .. Then the strips were subjected to a two-step equilibration in buffers: the first step containing 6 M urea, 30% glycerol, 2% SDS, and 50 mM Tris-HCl (pH 8.8) with 1% w/v dithiothreitol (DTT, Affymetrix/USB, 15395) and for the second step, with 2.5% w/v iodoacetamide (IAA, Amersham Biosciences, RPN6302V).

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    The native human Prp19/CDC5L complex contains four copies of hPrp19. (A) Determination of the protein stoichiometry by fluorescence staining. Four different amounts of hPrp19/CDC5L complex proteins (as indicated above each lane) were separated by SDS gel electrophoresis and stained with Sypro-Ruby. The fluorescence was detected by fluorescence imaging (FLA-7000; Fujifilm). (B) Determination of the protein stoichiometry by radioactive labeling. Denatured proteins (0.5 to 12.5 μg, as indicated) were labeled with [ 14 <t>C]iodoacetamide,</t> separated by SDS-PAGE and detected by autoradiography. (C) Band intensities obtained after Sypro-Ruby staining and [ 14 C]iodoacetamide labeling were quantified using Fuji Image Gauge v4.22 and normalized against the CDC5L signal. (D) Continuous size distribution [C (S) ] analysis of the hPrp19/CDC5L complex. Four different concentrations of the complex were analyzed (optical densities at 280 nm [OD 280 ] of 1.2, 0.6, 0.3, and 0.15) as indicated in the figure. The calculated C (S) is plotted against the sedimentation coefficient (S). The S values in the panel are corrected according to standard conditions [S (20,w) ]. (E) Multispeed sedimentation equilibrium analysis of the hPrp19/CDC5L complex at two different rotor speeds (gray, 5,500 rpm; black, 3,500 rpm). The OD 280 values along the cell were recorded and are shown in the upper panel. The lower panel shows the residuals between the fit (straight line in the upper panel) and the raw data (dots in the upper panel).
    C Iodoacetamide, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    GE Healthcare bodipy iam fluorescent signal intensity
    Oxidative modifications to proteins in Postoperative PCF A) Representative in-gel fluorescence image of individual patient PCF (0 and 4 hr) treated with 500 μM <t>Bodipy-iodoacetamide</t> <t>(Bodipy-IAM)</t> for 30 min to assess protein thiol modifications, where lower fluorescence intensity represents increased oxidized protein thiols. Following alkylation, 5 μg of protein was resolved by 12.5% SDS–PAGE and imaged in-gel using a Typhoon imager. The bottom panel shows coomassie blue stain to confirm equal protein loading. Quantitation of the Bodipy-IAM fluorescence signal for albumin normalized to coomassie blue protein staining. n=7 patients; Data expressed as mean ± SEM. * = p
    Bodipy Iam Fluorescent Signal Intensity, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    Image Search Results


    The native human Prp19/CDC5L complex contains four copies of hPrp19. (A) Determination of the protein stoichiometry by fluorescence staining. Four different amounts of hPrp19/CDC5L complex proteins (as indicated above each lane) were separated by SDS gel electrophoresis and stained with Sypro-Ruby. The fluorescence was detected by fluorescence imaging (FLA-7000; Fujifilm). (B) Determination of the protein stoichiometry by radioactive labeling. Denatured proteins (0.5 to 12.5 μg, as indicated) were labeled with [ 14 C]iodoacetamide, separated by SDS-PAGE and detected by autoradiography. (C) Band intensities obtained after Sypro-Ruby staining and [ 14 C]iodoacetamide labeling were quantified using Fuji Image Gauge v4.22 and normalized against the CDC5L signal. (D) Continuous size distribution [C (S) ] analysis of the hPrp19/CDC5L complex. Four different concentrations of the complex were analyzed (optical densities at 280 nm [OD 280 ] of 1.2, 0.6, 0.3, and 0.15) as indicated in the figure. The calculated C (S) is plotted against the sedimentation coefficient (S). The S values in the panel are corrected according to standard conditions [S (20,w) ]. (E) Multispeed sedimentation equilibrium analysis of the hPrp19/CDC5L complex at two different rotor speeds (gray, 5,500 rpm; black, 3,500 rpm). The OD 280 values along the cell were recorded and are shown in the upper panel. The lower panel shows the residuals between the fit (straight line in the upper panel) and the raw data (dots in the upper panel).

    Journal: Molecular and Cellular Biology

    Article Title: Molecular Architecture of the Human Prp19/CDC5L Complex ▿Molecular Architecture of the Human Prp19/CDC5L Complex ▿ †

    doi: 10.1128/MCB.01505-09

    Figure Lengend Snippet: The native human Prp19/CDC5L complex contains four copies of hPrp19. (A) Determination of the protein stoichiometry by fluorescence staining. Four different amounts of hPrp19/CDC5L complex proteins (as indicated above each lane) were separated by SDS gel electrophoresis and stained with Sypro-Ruby. The fluorescence was detected by fluorescence imaging (FLA-7000; Fujifilm). (B) Determination of the protein stoichiometry by radioactive labeling. Denatured proteins (0.5 to 12.5 μg, as indicated) were labeled with [ 14 C]iodoacetamide, separated by SDS-PAGE and detected by autoradiography. (C) Band intensities obtained after Sypro-Ruby staining and [ 14 C]iodoacetamide labeling were quantified using Fuji Image Gauge v4.22 and normalized against the CDC5L signal. (D) Continuous size distribution [C (S) ] analysis of the hPrp19/CDC5L complex. Four different concentrations of the complex were analyzed (optical densities at 280 nm [OD 280 ] of 1.2, 0.6, 0.3, and 0.15) as indicated in the figure. The calculated C (S) is plotted against the sedimentation coefficient (S). The S values in the panel are corrected according to standard conditions [S (20,w) ]. (E) Multispeed sedimentation equilibrium analysis of the hPrp19/CDC5L complex at two different rotor speeds (gray, 5,500 rpm; black, 3,500 rpm). The OD 280 values along the cell were recorded and are shown in the upper panel. The lower panel shows the residuals between the fit (straight line in the upper panel) and the raw data (dots in the upper panel).

    Article Snippet: Subsequently, the pH was increased to 9.0 with 100 mM Tris-HCl buffer, and [14 C]iodoacetamide (56 mCi/mmol; GE Healthcare) was added to a concentration of 50 mM.

    Techniques: Fluorescence, Staining, SDS-Gel, Electrophoresis, Imaging, Labeling, SDS Page, Autoradiography, Sedimentation

    The disulfide bridge in the Cys-rich motif affects the SCHIC-heme interaction. (A) A disulfide bridge was identified within peptide AppA 397-409 . Under oxidized conditions, neither Cys 399 nor Cys 406 can be modified by iodoacetamide (IAM). Under reduced conditions, the disulfide bridge formed between Cys 399 and Cys 406 is reduced, and both residues can be modified by iodoacetamide (DTT-IAM). (B) LC-MS-MS with double-labeled AppA identified the Cys 399 -Cys 406 disulfide bond (see Fig. S2 in the supplemental material). (C) The cysteine residues in AppA are reduced by DTT (AppA R ). When light excited, the Soret peak of AppA R -heme is redshifted from 412 nm to 418 nm. (D) When light excited, the Soret peak of AppA C399A -heme is redshifted from 412 nm to 418 nm.

    Journal: mBio

    Article Title: Redox and Light Control the Heme-Sensing Activity of AppA

    doi: 10.1128/mBio.00563-13

    Figure Lengend Snippet: The disulfide bridge in the Cys-rich motif affects the SCHIC-heme interaction. (A) A disulfide bridge was identified within peptide AppA 397-409 . Under oxidized conditions, neither Cys 399 nor Cys 406 can be modified by iodoacetamide (IAM). Under reduced conditions, the disulfide bridge formed between Cys 399 and Cys 406 is reduced, and both residues can be modified by iodoacetamide (DTT-IAM). (B) LC-MS-MS with double-labeled AppA identified the Cys 399 -Cys 406 disulfide bond (see Fig. S2 in the supplemental material). (C) The cysteine residues in AppA are reduced by DTT (AppA R ). When light excited, the Soret peak of AppA R -heme is redshifted from 412 nm to 418 nm. (D) When light excited, the Soret peak of AppA C399A -heme is redshifted from 412 nm to 418 nm.

    Article Snippet: Excess iodoacetamide was then removed by passing through a MidiTrap G-25 (GE Healthcare) desalting column or a Zeba spin desalting column with a molecular weight cutoff of 7,000 (7K MWCO) (Thermo Scientific), depending on the sample volume.

    Techniques: Modification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Labeling

    Oxidative modifications to proteins in Postoperative PCF A) Representative in-gel fluorescence image of individual patient PCF (0 and 4 hr) treated with 500 μM Bodipy-iodoacetamide (Bodipy-IAM) for 30 min to assess protein thiol modifications, where lower fluorescence intensity represents increased oxidized protein thiols. Following alkylation, 5 μg of protein was resolved by 12.5% SDS–PAGE and imaged in-gel using a Typhoon imager. The bottom panel shows coomassie blue stain to confirm equal protein loading. Quantitation of the Bodipy-IAM fluorescence signal for albumin normalized to coomassie blue protein staining. n=7 patients; Data expressed as mean ± SEM. * = p

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Hemoglobin-associated Oxidative Stress in the Pericardial Compartment of Post-operative Cardiac Surgery Patients

    doi: 10.1038/labinvest.2014.144

    Figure Lengend Snippet: Oxidative modifications to proteins in Postoperative PCF A) Representative in-gel fluorescence image of individual patient PCF (0 and 4 hr) treated with 500 μM Bodipy-iodoacetamide (Bodipy-IAM) for 30 min to assess protein thiol modifications, where lower fluorescence intensity represents increased oxidized protein thiols. Following alkylation, 5 μg of protein was resolved by 12.5% SDS–PAGE and imaged in-gel using a Typhoon imager. The bottom panel shows coomassie blue stain to confirm equal protein loading. Quantitation of the Bodipy-IAM fluorescence signal for albumin normalized to coomassie blue protein staining. n=7 patients; Data expressed as mean ± SEM. * = p

    Article Snippet: The Bodipy-IAM fluorescent signal intensity for albumin was quantified using ImageQuantTL analysis software (GE Healthcare Biosciences, Pittsburgh, PA) and the coomassie blue protein stain for albumin was quantified using the AlphaView SA software.

    Techniques: Fluorescence, SDS Page, Staining, Quantitation Assay