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Applichem hepes
Schematic Workflow for the DTR versus FASP comparison. Four single, deparaffinized murine kidney FFPE tissues were separately prepared with each of the three sample preparation protocol. For the DTR protocol, a buffer containing 0.1% <t>Rapigest</t> in 0.1 M <t>HEPES</t> pH 8 and 1 mM DTT was used for heat-induced antigen retrieval (HIAR) and lysis of the tissue. As RapiGest is compatible with tryptic digestion, direct trypsinization is the key feature of the DTR protocol. RapiGest is later removed by acidifying the sample. Protein concentration is estimated to not overload the C18 stage tips during desalting step and later to inject the same amounts of peptides into the mass spectrometer. The FASP protocol makes use of a buffer containing 4% SDS, 0.1 M HEPES pH 7.5 and 0.05 M DTT. Before digestion the SDS is removed using centrifugal filter units with nominal molecular weight cut offs of either 10,000 or 30,000 Da. After digestion, the peptides are eluted from the filter units and desalted before mass-spectrometry analysis
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1) Product Images from "Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization"

Article Title: Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization

Journal: Clinical Proteomics

doi: 10.1186/s12014-018-9188-y

Schematic Workflow for the DTR versus FASP comparison. Four single, deparaffinized murine kidney FFPE tissues were separately prepared with each of the three sample preparation protocol. For the DTR protocol, a buffer containing 0.1% Rapigest in 0.1 M HEPES pH 8 and 1 mM DTT was used for heat-induced antigen retrieval (HIAR) and lysis of the tissue. As RapiGest is compatible with tryptic digestion, direct trypsinization is the key feature of the DTR protocol. RapiGest is later removed by acidifying the sample. Protein concentration is estimated to not overload the C18 stage tips during desalting step and later to inject the same amounts of peptides into the mass spectrometer. The FASP protocol makes use of a buffer containing 4% SDS, 0.1 M HEPES pH 7.5 and 0.05 M DTT. Before digestion the SDS is removed using centrifugal filter units with nominal molecular weight cut offs of either 10,000 or 30,000 Da. After digestion, the peptides are eluted from the filter units and desalted before mass-spectrometry analysis
Figure Legend Snippet: Schematic Workflow for the DTR versus FASP comparison. Four single, deparaffinized murine kidney FFPE tissues were separately prepared with each of the three sample preparation protocol. For the DTR protocol, a buffer containing 0.1% Rapigest in 0.1 M HEPES pH 8 and 1 mM DTT was used for heat-induced antigen retrieval (HIAR) and lysis of the tissue. As RapiGest is compatible with tryptic digestion, direct trypsinization is the key feature of the DTR protocol. RapiGest is later removed by acidifying the sample. Protein concentration is estimated to not overload the C18 stage tips during desalting step and later to inject the same amounts of peptides into the mass spectrometer. The FASP protocol makes use of a buffer containing 4% SDS, 0.1 M HEPES pH 7.5 and 0.05 M DTT. Before digestion the SDS is removed using centrifugal filter units with nominal molecular weight cut offs of either 10,000 or 30,000 Da. After digestion, the peptides are eluted from the filter units and desalted before mass-spectrometry analysis

Techniques Used: Formalin-fixed Paraffin-Embedded, Sample Prep, Lysis, Protein Concentration, Mass Spectrometry, Molecular Weight

2) Product Images from "Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization"

Article Title: Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization

Journal: Clinical Proteomics

doi: 10.1186/s12014-018-9188-y

Schematic Workflow for the DTR versus FASP comparison. Four single, deparaffinized murine kidney FFPE tissues were separately prepared with each of the three sample preparation protocol. For the DTR protocol, a buffer containing 0.1% Rapigest in 0.1 M HEPES pH 8 and 1 mM DTT was used for heat-induced antigen retrieval (HIAR) and lysis of the tissue. As RapiGest is compatible with tryptic digestion, direct trypsinization is the key feature of the DTR protocol. RapiGest is later removed by acidifying the sample. Protein concentration is estimated to not overload the C18 stage tips during desalting step and later to inject the same amounts of peptides into the mass spectrometer. The FASP protocol makes use of a buffer containing 4% SDS, 0.1 M HEPES pH 7.5 and 0.05 M DTT. Before digestion the SDS is removed using centrifugal filter units with nominal molecular weight cut offs of either 10,000 or 30,000 Da. After digestion, the peptides are eluted from the filter units and desalted before mass-spectrometry analysis
Figure Legend Snippet: Schematic Workflow for the DTR versus FASP comparison. Four single, deparaffinized murine kidney FFPE tissues were separately prepared with each of the three sample preparation protocol. For the DTR protocol, a buffer containing 0.1% Rapigest in 0.1 M HEPES pH 8 and 1 mM DTT was used for heat-induced antigen retrieval (HIAR) and lysis of the tissue. As RapiGest is compatible with tryptic digestion, direct trypsinization is the key feature of the DTR protocol. RapiGest is later removed by acidifying the sample. Protein concentration is estimated to not overload the C18 stage tips during desalting step and later to inject the same amounts of peptides into the mass spectrometer. The FASP protocol makes use of a buffer containing 4% SDS, 0.1 M HEPES pH 7.5 and 0.05 M DTT. Before digestion the SDS is removed using centrifugal filter units with nominal molecular weight cut offs of either 10,000 or 30,000 Da. After digestion, the peptides are eluted from the filter units and desalted before mass-spectrometry analysis

Techniques Used: Formalin-fixed Paraffin-Embedded, Sample Prep, Lysis, Protein Concentration, Mass Spectrometry, Molecular Weight

Related Articles

Filtration:

Article Title: Comparison of drug release from liquid crystalline monoolein dispersions and solid lipid nanoparticles using a flow cytometric technique
Article Snippet: 2.1 Materials Triglycerides trimyristin (D114, Dynasan 114) and Miglyol 812 were from Condea Chemie (D-Witten), Poloxamer 407 (Lutrol F127) was from BASF AG (D-Ludwigshafen), sodium glycocholate (SGC) and 5,10,15,20-tetrakis (4-hydroxyphenyl)-21H , 23H -porphine (porphyrin) were from Sigma-Aldrich (D-Steinheim), Lipoid S75 was from Lipoid GmbH (D-Ludwigshafen), monoolein (GMOrphic-801) from Eastman Chemical Company (Kingsport, TN), methanol was from Carl Roth GmbH (D-Karlsruhe), acetonitrile, ethanol and chloroform all from VWR International (D-Darmstadt), tetrahydrofurane (THF) was from Fisher Scientific (D-Nidderau), and Hepes and sodium chloride were from AppliChem GmbH (D-Darmstadt). .. Purified water was prepared by filtration and deionization/reverse osmosis (Milli RX 20, Millipore, D-Schwalbach).

Article Title: Comparative Study on the Suitability of Two Techniques for Measuring the Transfer of Lipophilic Drug Models from Lipid Nanoparticles to Lipophilic Acceptors
Article Snippet: The triglyceride trimyristin (D114, Dynasan 114) and Miglyol 812 were a gift of Condea Chemie (Witten, Germany), partially hydrolyzed poly (vinyl alcohol) (PVA; Mowiol 3-83) was from Clariant (Frankfurt/Main, Germany), sodium glycocholate (SGC), cholesterol, Trizma 7.4 pre-set crystals, sucrose, praseodym (ΙΙΙ)-chloride (PrCl3 ), and sodium azide were from Sigma-Aldrich (Steinheim, Germany), egg phosphatidyl choline (EPC) and Lipoid S75 (S75) were obtained from Lipoid GmbH (Ludwigshafen, Germany), Nile red was obtained from Acros Organics (Geel, Belgium), temoporfin (5,10,15,20-tetrakis (3-hydroxyphenyl) chlorin) was a kind gift from Biolitec AG, Jena, Germany, glycerol from Solvay GmbH (Rheinberg, Germany), thiomersal from Caesar and Loretz (Hilden, Germany), methanol and tris were from Carl Roth GmbH (Karlsruhe, Germany), acetonitrile, ethanol, and chloroform all from VWR International (Darmstadt, Germany), tetrahydrofurane (THF) was from Fisher Scientific (Nidderau, Germany), and Hepes from AppliChem GmbH (Darmstadt, Germany). .. Purified water was prepared by filtration and deionization/reverse osmosis (Milli RX 20, Millipore, Schwalbach, Germany).

Positive Control:

Article Title: Antimicrobial Activity of α-Peptide/β-Peptoid Lysine-Based Peptidomimetics Against Colistin-Resistant Pseudomonas aeruginosa Isolated From Cystic Fibrosis Patients
Article Snippet: Briefly, the adhered cells were washed with 37°C Hanks’ balanced salt solution (HBSS from Sigma-Aldrich, St. Louis, MO, United States) containing 10 mM HEPES (AppliChem, Darmstadt, Germany), pH 7.4, and exposed for 1 h at 37°C to 100 μL of peptidomimetic dissolved in the medium also used for culturing of each cell line (at concentrations in the range 0–1000 μM). .. The relative viability was calculated by using 0.2% (w/v) sodium dodecyl sulfate (SDS) as the positive control, while cells exposed to medium without test compound were used as the negative control.

Synthesized:

Article Title: NOP receptor pharmacological profile – A dynamic mass redistribution study
Article Snippet: The non-peptide molecules Ro 65–6570, C-24, and J-113397 were synthesized in our laboratories. .. Hanks balanced salt solution (HBSS) was from Invitrogen (Darmstadt, DE), HEPES was from Applichem (Darmstadt, DE).

Incubation:

Article Title: Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization
Article Snippet: 100 µl (200 µl for the tonsil samples) of an aqueous buffer containing 0.1% RapiGest SF (Waters, Milford, MA, USA), 0.1 M HEPES pH 8 (AppliChem, Darmstadt, Germany) and 1 mM dithiothreitol (DTT) (AppliChem, Darmstadt, Germany) were added to each reaction tube, containing a single tissue slice. .. The buffered samples were incubated in a thermo shaker (TS1 ThermoShaker, Biometra, Göttingen, Germany) at 95 °C and 750 rpm for 4 h to perform heat induced antigen retrieval and protein extraction.

Article Title: Repurposing Azithromycin and Rifampicin Against Gram-Negative Pathogens by Combination With Peptidomimetics
Article Snippet: Briefly, the adhered cells were washed with 37°C Hanks' balanced salt solution (HBSS from Sigma-Aldrich, St. Louis, MO, USA), containing 10 mM HEPES (AppliChem, Darmstadt, Germany), and adjusted to pH 7.4, and were then exposed for 1 h to 100 μL of test compound(s) dissolved in the appropriate culturing medium without serum for each cell line. .. After exposure the cells were washed with HBSS containing 10 mM HEPES (pH 7.4) and 100 μL of an MTS/PMS solution, consisting of 240 μg/mL MTS (Promega, Madison, WI, USA) and 2.4 μg/mL PMS (SigmaAldrich, Buchs, Switzerland) in HBSS, was added to the cells, which then were incubated for 1 h at 37°C with horizontal shaking under light protection.

Article Title: Antimicrobial Activity of α-Peptide/β-Peptoid Lysine-Based Peptidomimetics Against Colistin-Resistant Pseudomonas aeruginosa Isolated From Cystic Fibrosis Patients
Article Snippet: Briefly, the adhered cells were washed with 37°C Hanks’ balanced salt solution (HBSS from Sigma-Aldrich, St. Louis, MO, United States) containing 10 mM HEPES (AppliChem, Darmstadt, Germany), pH 7.4, and exposed for 1 h at 37°C to 100 μL of peptidomimetic dissolved in the medium also used for culturing of each cell line (at concentrations in the range 0–1000 μM). .. Then the cells were washed twice with 37°C HBSS containing 10 mM HEPES (pH 7.4), and then 100 μL of an MTS/PMS solution, consisting of 240 μg/mL MTS (Promega, Madison, WI, United States) and 2.4 mg/mL PMS (Sigma-Aldrich, Buchs, Switzerland) in HBSS, were added to the cells, which then were incubated for 1 h at 37°C with horizontal shaking (50 rpm) protected from light.

Article Title: Rapid detection of 2-hydroxyglutarate in frozen sections of IDH mutant tumors by MALDI-TOF mass spectrometry
Article Snippet: The assay solution contained 100 mM HEPES pH 8.0, 100 μM NAD+ , 5 μM resazurin (Applichem, Darmstadt, Germany), 0.1 μg HGDH and 0.01 U/ml diaphorase (0.01 U/ml; MP Biomedical, Irvine, USA). .. Immediately before use, 25 μl sample volume was added to 75 μl of assay solution and incubated at room temperature for 30 min in black 96-well plates (Thermo Fisher Scientific, Waltham, USA) in the dark.

Activity Assay:

Article Title: Repurposing Azithromycin and Rifampicin Against Gram-Negative Pathogens by Combination With Peptidomimetics
Article Snippet: Effect on cell viability was determined in NIH 3T3 fibroblasts and HepG2 hepatocytes by using the MTS/PMS assay measuring metabolic activity. .. Briefly, the adhered cells were washed with 37°C Hanks' balanced salt solution (HBSS from Sigma-Aldrich, St. Louis, MO, USA), containing 10 mM HEPES (AppliChem, Darmstadt, Germany), and adjusted to pH 7.4, and were then exposed for 1 h to 100 μL of test compound(s) dissolved in the appropriate culturing medium without serum for each cell line.

Expressing:

Article Title: Affinity Map of Bromodomain Protein 4 (BRD4) Interactions with the Histone H4 Tail and the Small Molecule Inhibitor JQ1 *
Article Snippet: .. Bacteria expressing His6 -tagged proteins BRD4 BD1 or BD2 wild type were resuspended in 50 mm HEPES, pH 7.5 (Applichem), 500 mm NaCl (Sigma), 5% glycerol (Sigma), 5 mm imidazole (Sigma), 0.5 mm Tris(2-carboxyethyl)phosphine (Pierce), and EDTA-free Complete, and lysed using a Microfluidizer (Microfluidics). .. The proteins were then purified by affinity chromatography using nickel-nitrilotriacetic acid-agarose (Macherey-Nagel) or His-trap columns (GE Healthcare) followed by a size exclusion chromatography step using a Superdex S75 26/60 column (GE Healthcare).

Modification:

Article Title: Repurposing Azithromycin and Rifampicin Against Gram-Negative Pathogens by Combination With Peptidomimetics
Article Snippet: NIH 3T3 were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) newborn-calf serum (NCS) (Gibco, Paisly, UK), while HepG2 were cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, Paisly, UK), sodium pyruvate (1 mM), and non-essential amino acids (1%, v/v). .. Briefly, the adhered cells were washed with 37°C Hanks' balanced salt solution (HBSS from Sigma-Aldrich, St. Louis, MO, USA), containing 10 mM HEPES (AppliChem, Darmstadt, Germany), and adjusted to pH 7.4, and were then exposed for 1 h to 100 μL of test compound(s) dissolved in the appropriate culturing medium without serum for each cell line.

Article Title: Cyclin-dependent kinases 4 and 6 control tumor progression and direct glucose oxidation in the pentose cycle
Article Snippet: .. Dulbecco’s modified Eagle Medium (DMEM), F-12 HAM Nutrient mixture with L-glutamine, MEM-EAGLE non-essential aminoacid solution ×100, antibiotic (100 U/ml penicillin, 100 mg/ml streptomycin), Dulbecco’s Phosphate buffer saline (PBS), Trypsin EDTA solution C (0.05% trypsin–0.02% EDTA), L-glutamine solution 200 mM and sodium pyruvate solution 100 mM were obtained from Biological Industries; Fetal calf serum (FCS) and Trizol were from Invitrogen; SDS was from Fluka; Coomassie blue was from Biorad; HEPES and MgCl2 were from Applichem; A-Sepharose was from Pierce; the [ γ -32P]ATP, 3000 Ci/mmol, 10 mCi/ml and ECL were from Amersham; histone H1 was from Boehringer Mannheim; Bradford reagent (500-0006), Acrylamide (161-0158) and peroxidase-coupled secondary antibody were from Bio-Rad Laboratories; anti-CDK6 (sc-177), anti-CDK4 (sc-260-R), anti-cyclin D3 (sc-182) and anti-p16INK4a (sc-468) were from Santa Cruz Biotechnology; anti-cyclin D1 (06-137), anti-CDK2 (06-505) and anti-cyclin B1 (05-158) were from Upstate Biotechnology; anti-actin (691001) was from MP Biomedicals; anti-phospho-Rb (Ser780) was from Cell Signaling Technology; pGST-Rb (379–928) (gift of Dr Wang, San Diego, CA, USA) fusion protein was expressed and purified following and . ..

Article Title: ER–mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem‐like cells
Article Snippet: .. GL261 and U251 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco) with 10% fetal bovine serum (FBS) (Gibco) and supplements (100 U/ml penicillin G (Sigma‐Aldrich), 100 μg/ml streptomycin sulfate (Sigma‐Aldrich), 6 mM Hepes (Applichem), 1.6 mM l ‐glutamine (Sigma‐Aldrich), 50 μM β‐mercaptoethanol (Bio‐Rad). .. GE904 were isolated from one patient biopsy and directly grown in the same medium supplemented with none‐essential amino acids.

Cell Culture:

Article Title: Repurposing Azithromycin and Rifampicin Against Gram-Negative Pathogens by Combination With Peptidomimetics
Article Snippet: NIH 3T3 were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) newborn-calf serum (NCS) (Gibco, Paisly, UK), while HepG2 were cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, Paisly, UK), sodium pyruvate (1 mM), and non-essential amino acids (1%, v/v). .. Briefly, the adhered cells were washed with 37°C Hanks' balanced salt solution (HBSS from Sigma-Aldrich, St. Louis, MO, USA), containing 10 mM HEPES (AppliChem, Darmstadt, Germany), and adjusted to pH 7.4, and were then exposed for 1 h to 100 μL of test compound(s) dissolved in the appropriate culturing medium without serum for each cell line.

Article Title: CD28 Costimulation of T Helper 1 Cells Enhances Cytokine Release In Vivo
Article Snippet: .. Isolated carboxyfluorescein succinimidyl ester diacetate (CFSE) (5 µM) labeled PBMCs were cultured in RPMI 1640 medium supplemented with l -glutamine (Invitrogen), nonessential amino acids (Invitrogen), HEPES (Applichem), β-mercaptoethanol (Invitrogen), sodium pyruvate (Invitrogen), penicillin/streptomycin, and 10% heat-inactivated human AB serum (Sigma-Aldrich) in the presence or absence of 0.1 µg/ml anti-CD3 mAb (HIT3a), 10 µg/ml purified protein derivative (PPD) (Pharmore), 100 mU/ml tetanus and diphtheria toxoid (Td)-RIX (GlaxoSmithKline), and 0.3 µg/ml Fab fragment of the anti-human CD28 mAb CD28.3. .. To generate Th1 conditions, 1 µg/ml anti-human IL-4 (R & D Systems), 2 ng/ml rhIL-12 (Sigma) and, additionally, 0.1 µM rhIL-2 (Proleukin® , Novartis) were added ( ).

Article Title: ER–mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem‐like cells
Article Snippet: Paragraph title: Cell culture and reagents ... GL261 and U251 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco) with 10% fetal bovine serum (FBS) (Gibco) and supplements (100 U/ml penicillin G (Sigma‐Aldrich), 100 μg/ml streptomycin sulfate (Sigma‐Aldrich), 6 mM Hepes (Applichem), 1.6 mM l ‐glutamine (Sigma‐Aldrich), 50 μM β‐mercaptoethanol (Bio‐Rad).

Article Title: Self-Recognition Sensitizes Mouse and Human Regulatory T Cells to Low-Dose CD28 Superagonist Stimulation
Article Snippet: .. Cell Culture and Stimulation Assays (Human) Cells were cultured in RPMI 1640 supplemented with l -glutamine (Gibco), non-essential amino acids (Gibco), HEPES (Applichem), β-mercaptoethanol (Gibco), sodium pyruvate (Gibco), penicillin/streptomycin, and 10% AB-positive heat-inactivated human serum (Sigma) (AB medium). .. PBMC were first cultured for 2 days at a high cell density (1 × 107 /ml) in AB medium to reset them to tissue-like conditions without changing their cellular composition, thereby allowing reactivity to TGN1412/TAB08 in the secondary cultures.

other:

Article Title: System immunology-based identification of blood transcriptional modules correlating to antibody responses in sheep
Article Snippet: DPPC, DCChol, and PHAD were purchased from Avanti Polar Lipids; Hepes from Applichem; sucrose, chloroform, and ethanol from Sigma-Aldrich (Switzerland).

Sequencing:

Article Title: Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization
Article Snippet: 100 µl (200 µl for the tonsil samples) of an aqueous buffer containing 0.1% RapiGest SF (Waters, Milford, MA, USA), 0.1 M HEPES pH 8 (AppliChem, Darmstadt, Germany) and 1 mM dithiothreitol (DTT) (AppliChem, Darmstadt, Germany) were added to each reaction tube, containing a single tissue slice. .. Sequencing grade trypsin (Worthington, Lakewood, NJ, USA) was added in a ratio of at least 2 µg per mm3 tissue.

Isolation:

Article Title: CD28 Costimulation of T Helper 1 Cells Enhances Cytokine Release In Vivo
Article Snippet: .. Isolated carboxyfluorescein succinimidyl ester diacetate (CFSE) (5 µM) labeled PBMCs were cultured in RPMI 1640 medium supplemented with l -glutamine (Invitrogen), nonessential amino acids (Invitrogen), HEPES (Applichem), β-mercaptoethanol (Invitrogen), sodium pyruvate (Invitrogen), penicillin/streptomycin, and 10% heat-inactivated human AB serum (Sigma-Aldrich) in the presence or absence of 0.1 µg/ml anti-CD3 mAb (HIT3a), 10 µg/ml purified protein derivative (PPD) (Pharmore), 100 mU/ml tetanus and diphtheria toxoid (Td)-RIX (GlaxoSmithKline), and 0.3 µg/ml Fab fragment of the anti-human CD28 mAb CD28.3. .. To generate Th1 conditions, 1 µg/ml anti-human IL-4 (R & D Systems), 2 ng/ml rhIL-12 (Sigma) and, additionally, 0.1 µM rhIL-2 (Proleukin® , Novartis) were added ( ).

Article Title: ER–mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem‐like cells
Article Snippet: GL261 and U251 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco) with 10% fetal bovine serum (FBS) (Gibco) and supplements (100 U/ml penicillin G (Sigma‐Aldrich), 100 μg/ml streptomycin sulfate (Sigma‐Aldrich), 6 mM Hepes (Applichem), 1.6 mM l ‐glutamine (Sigma‐Aldrich), 50 μM β‐mercaptoethanol (Bio‐Rad). .. GE904 were isolated from one patient biopsy and directly grown in the same medium supplemented with none‐essential amino acids.

Size-exclusion Chromatography:

Article Title: Affinity Map of Bromodomain Protein 4 (BRD4) Interactions with the Histone H4 Tail and the Small Molecule Inhibitor JQ1 *
Article Snippet: Bacteria expressing His6 -tagged proteins BRD4 BD1 or BD2 wild type were resuspended in 50 mm HEPES, pH 7.5 (Applichem), 500 mm NaCl (Sigma), 5% glycerol (Sigma), 5 mm imidazole (Sigma), 0.5 mm Tris(2-carboxyethyl)phosphine (Pierce), and EDTA-free Complete, and lysed using a Microfluidizer (Microfluidics). .. The proteins were then purified by affinity chromatography using nickel-nitrilotriacetic acid-agarose (Macherey-Nagel) or His-trap columns (GE Healthcare) followed by a size exclusion chromatography step using a Superdex S75 26/60 column (GE Healthcare).

Labeling:

Article Title: CD28 Costimulation of T Helper 1 Cells Enhances Cytokine Release In Vivo
Article Snippet: .. Isolated carboxyfluorescein succinimidyl ester diacetate (CFSE) (5 µM) labeled PBMCs were cultured in RPMI 1640 medium supplemented with l -glutamine (Invitrogen), nonessential amino acids (Invitrogen), HEPES (Applichem), β-mercaptoethanol (Invitrogen), sodium pyruvate (Invitrogen), penicillin/streptomycin, and 10% heat-inactivated human AB serum (Sigma-Aldrich) in the presence or absence of 0.1 µg/ml anti-CD3 mAb (HIT3a), 10 µg/ml purified protein derivative (PPD) (Pharmore), 100 mU/ml tetanus and diphtheria toxoid (Td)-RIX (GlaxoSmithKline), and 0.3 µg/ml Fab fragment of the anti-human CD28 mAb CD28.3. .. To generate Th1 conditions, 1 µg/ml anti-human IL-4 (R & D Systems), 2 ng/ml rhIL-12 (Sigma) and, additionally, 0.1 µM rhIL-2 (Proleukin® , Novartis) were added ( ).

Purification:

Article Title: Affinity Map of Bromodomain Protein 4 (BRD4) Interactions with the Histone H4 Tail and the Small Molecule Inhibitor JQ1 *
Article Snippet: Paragraph title: Protein Expression and Purification ... Bacteria expressing His6 -tagged proteins BRD4 BD1 or BD2 wild type were resuspended in 50 mm HEPES, pH 7.5 (Applichem), 500 mm NaCl (Sigma), 5% glycerol (Sigma), 5 mm imidazole (Sigma), 0.5 mm Tris(2-carboxyethyl)phosphine (Pierce), and EDTA-free Complete, and lysed using a Microfluidizer (Microfluidics).

Article Title: Comparison of drug release from liquid crystalline monoolein dispersions and solid lipid nanoparticles using a flow cytometric technique
Article Snippet: 2.1 Materials Triglycerides trimyristin (D114, Dynasan 114) and Miglyol 812 were from Condea Chemie (D-Witten), Poloxamer 407 (Lutrol F127) was from BASF AG (D-Ludwigshafen), sodium glycocholate (SGC) and 5,10,15,20-tetrakis (4-hydroxyphenyl)-21H , 23H -porphine (porphyrin) were from Sigma-Aldrich (D-Steinheim), Lipoid S75 was from Lipoid GmbH (D-Ludwigshafen), monoolein (GMOrphic-801) from Eastman Chemical Company (Kingsport, TN), methanol was from Carl Roth GmbH (D-Karlsruhe), acetonitrile, ethanol and chloroform all from VWR International (D-Darmstadt), tetrahydrofurane (THF) was from Fisher Scientific (D-Nidderau), and Hepes and sodium chloride were from AppliChem GmbH (D-Darmstadt). .. Purified water was prepared by filtration and deionization/reverse osmosis (Milli RX 20, Millipore, D-Schwalbach).

Article Title: CD28 Costimulation of T Helper 1 Cells Enhances Cytokine Release In Vivo
Article Snippet: .. Isolated carboxyfluorescein succinimidyl ester diacetate (CFSE) (5 µM) labeled PBMCs were cultured in RPMI 1640 medium supplemented with l -glutamine (Invitrogen), nonessential amino acids (Invitrogen), HEPES (Applichem), β-mercaptoethanol (Invitrogen), sodium pyruvate (Invitrogen), penicillin/streptomycin, and 10% heat-inactivated human AB serum (Sigma-Aldrich) in the presence or absence of 0.1 µg/ml anti-CD3 mAb (HIT3a), 10 µg/ml purified protein derivative (PPD) (Pharmore), 100 mU/ml tetanus and diphtheria toxoid (Td)-RIX (GlaxoSmithKline), and 0.3 µg/ml Fab fragment of the anti-human CD28 mAb CD28.3. .. To generate Th1 conditions, 1 µg/ml anti-human IL-4 (R & D Systems), 2 ng/ml rhIL-12 (Sigma) and, additionally, 0.1 µM rhIL-2 (Proleukin® , Novartis) were added ( ).

Article Title: Cyclin-dependent kinases 4 and 6 control tumor progression and direct glucose oxidation in the pentose cycle
Article Snippet: .. Dulbecco’s modified Eagle Medium (DMEM), F-12 HAM Nutrient mixture with L-glutamine, MEM-EAGLE non-essential aminoacid solution ×100, antibiotic (100 U/ml penicillin, 100 mg/ml streptomycin), Dulbecco’s Phosphate buffer saline (PBS), Trypsin EDTA solution C (0.05% trypsin–0.02% EDTA), L-glutamine solution 200 mM and sodium pyruvate solution 100 mM were obtained from Biological Industries; Fetal calf serum (FCS) and Trizol were from Invitrogen; SDS was from Fluka; Coomassie blue was from Biorad; HEPES and MgCl2 were from Applichem; A-Sepharose was from Pierce; the [ γ -32P]ATP, 3000 Ci/mmol, 10 mCi/ml and ECL were from Amersham; histone H1 was from Boehringer Mannheim; Bradford reagent (500-0006), Acrylamide (161-0158) and peroxidase-coupled secondary antibody were from Bio-Rad Laboratories; anti-CDK6 (sc-177), anti-CDK4 (sc-260-R), anti-cyclin D3 (sc-182) and anti-p16INK4a (sc-468) were from Santa Cruz Biotechnology; anti-cyclin D1 (06-137), anti-CDK2 (06-505) and anti-cyclin B1 (05-158) were from Upstate Biotechnology; anti-actin (691001) was from MP Biomedicals; anti-phospho-Rb (Ser780) was from Cell Signaling Technology; pGST-Rb (379–928) (gift of Dr Wang, San Diego, CA, USA) fusion protein was expressed and purified following and . ..

Article Title: Comparative Study on the Suitability of Two Techniques for Measuring the Transfer of Lipophilic Drug Models from Lipid Nanoparticles to Lipophilic Acceptors
Article Snippet: The triglyceride trimyristin (D114, Dynasan 114) and Miglyol 812 were a gift of Condea Chemie (Witten, Germany), partially hydrolyzed poly (vinyl alcohol) (PVA; Mowiol 3-83) was from Clariant (Frankfurt/Main, Germany), sodium glycocholate (SGC), cholesterol, Trizma 7.4 pre-set crystals, sucrose, praseodym (ΙΙΙ)-chloride (PrCl3 ), and sodium azide were from Sigma-Aldrich (Steinheim, Germany), egg phosphatidyl choline (EPC) and Lipoid S75 (S75) were obtained from Lipoid GmbH (Ludwigshafen, Germany), Nile red was obtained from Acros Organics (Geel, Belgium), temoporfin (5,10,15,20-tetrakis (3-hydroxyphenyl) chlorin) was a kind gift from Biolitec AG, Jena, Germany, glycerol from Solvay GmbH (Rheinberg, Germany), thiomersal from Caesar and Loretz (Hilden, Germany), methanol and tris were from Carl Roth GmbH (Karlsruhe, Germany), acetonitrile, ethanol, and chloroform all from VWR International (Darmstadt, Germany), tetrahydrofurane (THF) was from Fisher Scientific (Nidderau, Germany), and Hepes from AppliChem GmbH (Darmstadt, Germany). .. Purified water was prepared by filtration and deionization/reverse osmosis (Milli RX 20, Millipore, Schwalbach, Germany).

Protein Extraction:

Article Title: Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization
Article Snippet: 100 µl (200 µl for the tonsil samples) of an aqueous buffer containing 0.1% RapiGest SF (Waters, Milford, MA, USA), 0.1 M HEPES pH 8 (AppliChem, Darmstadt, Germany) and 1 mM dithiothreitol (DTT) (AppliChem, Darmstadt, Germany) were added to each reaction tube, containing a single tissue slice. .. The buffered samples were incubated in a thermo shaker (TS1 ThermoShaker, Biometra, Göttingen, Germany) at 95 °C and 750 rpm for 4 h to perform heat induced antigen retrieval and protein extraction.

Staining:

Article Title: Selective ribosome profiling as a tool to study the interaction of chaperones and targeting factors with nascent polypeptide chains and ribosomes
Article Snippet: HEPES (AppliChem, A1069) MgCl2 (Roth, A537) Isopropanol (2-propanol) (Sigma, 33539) CAUTION Isopropanol is flammable. .. 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC, Thermo Scientific, 22980) Glycine (Roth, 3790) NaHCO3 (Roth, 6885) Superase•In RNase inhibitor (Ambion, AM2696) Micrococcal nuclease (Nuclease S7) (Roche, 10107921001 or ) Ethylene glycol tetraacetic acid (EGTA, AppliChem, A0878) Sucrose (Sigma, 16104) Strep-Tactin Sepharose 50% suspension (IBA, 2-1201-025) DNA, sodium salt from salmon testes (Sigma-Aldrich, D1626) Kanamycin (Applichem, A1493) Isopropyl β-D-1-thiogalactopyranoside (IPTG, Roth, CN08) Colloidal coomassie staining solution (Roth, Roti-Blue quick, 4829) Imidazole (Roth, 3899) CAUTION Imidazole is corrosive.

Viability Assay:

Article Title: Antimicrobial Activity of α-Peptide/β-Peptoid Lysine-Based Peptidomimetics Against Colistin-Resistant Pseudomonas aeruginosa Isolated From Cystic Fibrosis Patients
Article Snippet: Paragraph title: Cell Viability Assay ... Briefly, the adhered cells were washed with 37°C Hanks’ balanced salt solution (HBSS from Sigma-Aldrich, St. Louis, MO, United States) containing 10 mM HEPES (AppliChem, Darmstadt, Germany), pH 7.4, and exposed for 1 h at 37°C to 100 μL of peptidomimetic dissolved in the medium also used for culturing of each cell line (at concentrations in the range 0–1000 μM).

Software:

Article Title: Repurposing Azithromycin and Rifampicin Against Gram-Negative Pathogens by Combination With Peptidomimetics
Article Snippet: Briefly, the adhered cells were washed with 37°C Hanks' balanced salt solution (HBSS from Sigma-Aldrich, St. Louis, MO, USA), containing 10 mM HEPES (AppliChem, Darmstadt, Germany), and adjusted to pH 7.4, and were then exposed for 1 h to 100 μL of test compound(s) dissolved in the appropriate culturing medium without serum for each cell line. .. The relative viability was calculated according to Equation (1) with absorbance values obtained after incubation of cells with test compound; incubation with SDS (0.2%, w/v in medium) defined 100% cell death (Abspos ), while the absorbance of cells incubated with medium defined 0% cell death (Absneg ). (1) R e l a t i v e v i a b i l i t y ( % ) = ( A b s s a m p l e - A b s p o s ) ( A b s n e g - A b s p o s ) × 100 % EC50 values were calculated by using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA) via fitting of the relative cell viability to the concentration of the test compound by using Equation (2): (2) R e l a t i v e v i a b i l i t y ( % ) = T o p - B o t t o m 1 + 1 0 ( L o g I C 50 - L o g [ p e p t i d o m i m e t i c ] ) × H i l l s l o p e With top and bottom representing the mean highest and lowest observed values, respectively.

Negative Control:

Article Title: Antimicrobial Activity of α-Peptide/β-Peptoid Lysine-Based Peptidomimetics Against Colistin-Resistant Pseudomonas aeruginosa Isolated From Cystic Fibrosis Patients
Article Snippet: Briefly, the adhered cells were washed with 37°C Hanks’ balanced salt solution (HBSS from Sigma-Aldrich, St. Louis, MO, United States) containing 10 mM HEPES (AppliChem, Darmstadt, Germany), pH 7.4, and exposed for 1 h at 37°C to 100 μL of peptidomimetic dissolved in the medium also used for culturing of each cell line (at concentrations in the range 0–1000 μM). .. The relative viability was calculated by using 0.2% (w/v) sodium dodecyl sulfate (SDS) as the positive control, while cells exposed to medium without test compound were used as the negative control.

Affinity Chromatography:

Article Title: Affinity Map of Bromodomain Protein 4 (BRD4) Interactions with the Histone H4 Tail and the Small Molecule Inhibitor JQ1 *
Article Snippet: Bacteria expressing His6 -tagged proteins BRD4 BD1 or BD2 wild type were resuspended in 50 mm HEPES, pH 7.5 (Applichem), 500 mm NaCl (Sigma), 5% glycerol (Sigma), 5 mm imidazole (Sigma), 0.5 mm Tris(2-carboxyethyl)phosphine (Pierce), and EDTA-free Complete, and lysed using a Microfluidizer (Microfluidics). .. The proteins were then purified by affinity chromatography using nickel-nitrilotriacetic acid-agarose (Macherey-Nagel) or His-trap columns (GE Healthcare) followed by a size exclusion chromatography step using a Superdex S75 26/60 column (GE Healthcare).

Sample Prep:

Article Title: Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization
Article Snippet: Direct tissue trypsinization using a RapiGest containing buffer (DTR) The sample preparation steps for the DTR protocol are shown in Fig. . .. 100 µl (200 µl for the tonsil samples) of an aqueous buffer containing 0.1% RapiGest SF (Waters, Milford, MA, USA), 0.1 M HEPES pH 8 (AppliChem, Darmstadt, Germany) and 1 mM dithiothreitol (DTT) (AppliChem, Darmstadt, Germany) were added to each reaction tube, containing a single tissue slice.

In Vitro:

Article Title: CD28 Costimulation of T Helper 1 Cells Enhances Cytokine Release In Vivo
Article Snippet: Paragraph title: In Vitro Recall Responses (Human) ... Isolated carboxyfluorescein succinimidyl ester diacetate (CFSE) (5 µM) labeled PBMCs were cultured in RPMI 1640 medium supplemented with l -glutamine (Invitrogen), nonessential amino acids (Invitrogen), HEPES (Applichem), β-mercaptoethanol (Invitrogen), sodium pyruvate (Invitrogen), penicillin/streptomycin, and 10% heat-inactivated human AB serum (Sigma-Aldrich) in the presence or absence of 0.1 µg/ml anti-CD3 mAb (HIT3a), 10 µg/ml purified protein derivative (PPD) (Pharmore), 100 mU/ml tetanus and diphtheria toxoid (Td)-RIX (GlaxoSmithKline), and 0.3 µg/ml Fab fragment of the anti-human CD28 mAb CD28.3.

Concentration Assay:

Article Title: Repurposing Azithromycin and Rifampicin Against Gram-Negative Pathogens by Combination With Peptidomimetics
Article Snippet: Briefly, the adhered cells were washed with 37°C Hanks' balanced salt solution (HBSS from Sigma-Aldrich, St. Louis, MO, USA), containing 10 mM HEPES (AppliChem, Darmstadt, Germany), and adjusted to pH 7.4, and were then exposed for 1 h to 100 μL of test compound(s) dissolved in the appropriate culturing medium without serum for each cell line. .. The relative viability was calculated according to Equation (1) with absorbance values obtained after incubation of cells with test compound; incubation with SDS (0.2%, w/v in medium) defined 100% cell death (Abspos ), while the absorbance of cells incubated with medium defined 0% cell death (Absneg ). (1) R e l a t i v e v i a b i l i t y ( % ) = ( A b s s a m p l e - A b s p o s ) ( A b s n e g - A b s p o s ) × 100 % EC50 values were calculated by using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA) via fitting of the relative cell viability to the concentration of the test compound by using Equation (2): (2) R e l a t i v e v i a b i l i t y ( % ) = T o p - B o t t o m 1 + 1 0 ( L o g I C 50 - L o g [ p e p t i d o m i m e t i c ] ) × H i l l s l o p e With top and bottom representing the mean highest and lowest observed values, respectively.

Lysis:

Article Title: Rapid detection of 2-hydroxyglutarate in frozen sections of IDH mutant tumors by MALDI-TOF mass spectrometry
Article Snippet: In brief, three 10 μm-thick slices were dissolved in 125 μl cell lysis buffer (150 mM NaCl, 0.1% NP-40, 50 mM Tris-HCl, pH 8.0) and subsequently treated with a deproteinization kit (Biovision, Mountain View, CA, USA). .. The assay solution contained 100 mM HEPES pH 8.0, 100 μM NAD+ , 5 μM resazurin (Applichem, Darmstadt, Germany), 0.1 μg HGDH and 0.01 U/ml diaphorase (0.01 U/ml; MP Biomedical, Irvine, USA).

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    Applichem hepes
    Schematic Workflow for the DTR versus FASP comparison. Four single, deparaffinized murine kidney FFPE tissues were separately prepared with each of the three sample preparation protocol. For the DTR protocol, a buffer containing 0.1% <t>Rapigest</t> in 0.1 M <t>HEPES</t> pH 8 and 1 mM DTT was used for heat-induced antigen retrieval (HIAR) and lysis of the tissue. As RapiGest is compatible with tryptic digestion, direct trypsinization is the key feature of the DTR protocol. RapiGest is later removed by acidifying the sample. Protein concentration is estimated to not overload the C18 stage tips during desalting step and later to inject the same amounts of peptides into the mass spectrometer. The FASP protocol makes use of a buffer containing 4% SDS, 0.1 M HEPES pH 7.5 and 0.05 M DTT. Before digestion the SDS is removed using centrifugal filter units with nominal molecular weight cut offs of either 10,000 or 30,000 Da. After digestion, the peptides are eluted from the filter units and desalted before mass-spectrometry analysis
    Hepes, supplied by Applichem, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Schematic Workflow for the DTR versus FASP comparison. Four single, deparaffinized murine kidney FFPE tissues were separately prepared with each of the three sample preparation protocol. For the DTR protocol, a buffer containing 0.1% Rapigest in 0.1 M HEPES pH 8 and 1 mM DTT was used for heat-induced antigen retrieval (HIAR) and lysis of the tissue. As RapiGest is compatible with tryptic digestion, direct trypsinization is the key feature of the DTR protocol. RapiGest is later removed by acidifying the sample. Protein concentration is estimated to not overload the C18 stage tips during desalting step and later to inject the same amounts of peptides into the mass spectrometer. The FASP protocol makes use of a buffer containing 4% SDS, 0.1 M HEPES pH 7.5 and 0.05 M DTT. Before digestion the SDS is removed using centrifugal filter units with nominal molecular weight cut offs of either 10,000 or 30,000 Da. After digestion, the peptides are eluted from the filter units and desalted before mass-spectrometry analysis

    Journal: Clinical Proteomics

    Article Title: Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization

    doi: 10.1186/s12014-018-9188-y

    Figure Lengend Snippet: Schematic Workflow for the DTR versus FASP comparison. Four single, deparaffinized murine kidney FFPE tissues were separately prepared with each of the three sample preparation protocol. For the DTR protocol, a buffer containing 0.1% Rapigest in 0.1 M HEPES pH 8 and 1 mM DTT was used for heat-induced antigen retrieval (HIAR) and lysis of the tissue. As RapiGest is compatible with tryptic digestion, direct trypsinization is the key feature of the DTR protocol. RapiGest is later removed by acidifying the sample. Protein concentration is estimated to not overload the C18 stage tips during desalting step and later to inject the same amounts of peptides into the mass spectrometer. The FASP protocol makes use of a buffer containing 4% SDS, 0.1 M HEPES pH 7.5 and 0.05 M DTT. Before digestion the SDS is removed using centrifugal filter units with nominal molecular weight cut offs of either 10,000 or 30,000 Da. After digestion, the peptides are eluted from the filter units and desalted before mass-spectrometry analysis

    Article Snippet: 100 µl (200 µl for the tonsil samples) of an aqueous buffer containing 0.1% RapiGest SF (Waters, Milford, MA, USA), 0.1 M HEPES pH 8 (AppliChem, Darmstadt, Germany) and 1 mM dithiothreitol (DTT) (AppliChem, Darmstadt, Germany) were added to each reaction tube, containing a single tissue slice.

    Techniques: Formalin-fixed Paraffin-Embedded, Sample Prep, Lysis, Protein Concentration, Mass Spectrometry, Molecular Weight

    Schematic Workflow for the DTR versus FASP comparison. Four single, deparaffinized murine kidney FFPE tissues were separately prepared with each of the three sample preparation protocol. For the DTR protocol, a buffer containing 0.1% Rapigest in 0.1 M HEPES pH 8 and 1 mM DTT was used for heat-induced antigen retrieval (HIAR) and lysis of the tissue. As RapiGest is compatible with tryptic digestion, direct trypsinization is the key feature of the DTR protocol. RapiGest is later removed by acidifying the sample. Protein concentration is estimated to not overload the C18 stage tips during desalting step and later to inject the same amounts of peptides into the mass spectrometer. The FASP protocol makes use of a buffer containing 4% SDS, 0.1 M HEPES pH 7.5 and 0.05 M DTT. Before digestion the SDS is removed using centrifugal filter units with nominal molecular weight cut offs of either 10,000 or 30,000 Da. After digestion, the peptides are eluted from the filter units and desalted before mass-spectrometry analysis

    Journal: Clinical Proteomics

    Article Title: Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization

    doi: 10.1186/s12014-018-9188-y

    Figure Lengend Snippet: Schematic Workflow for the DTR versus FASP comparison. Four single, deparaffinized murine kidney FFPE tissues were separately prepared with each of the three sample preparation protocol. For the DTR protocol, a buffer containing 0.1% Rapigest in 0.1 M HEPES pH 8 and 1 mM DTT was used for heat-induced antigen retrieval (HIAR) and lysis of the tissue. As RapiGest is compatible with tryptic digestion, direct trypsinization is the key feature of the DTR protocol. RapiGest is later removed by acidifying the sample. Protein concentration is estimated to not overload the C18 stage tips during desalting step and later to inject the same amounts of peptides into the mass spectrometer. The FASP protocol makes use of a buffer containing 4% SDS, 0.1 M HEPES pH 7.5 and 0.05 M DTT. Before digestion the SDS is removed using centrifugal filter units with nominal molecular weight cut offs of either 10,000 or 30,000 Da. After digestion, the peptides are eluted from the filter units and desalted before mass-spectrometry analysis

    Article Snippet: 100 µl (200 µl for the tonsil samples) of an aqueous buffer containing 0.1% RapiGest SF (Waters, Milford, MA, USA), 0.1 M HEPES pH 8 (AppliChem, Darmstadt, Germany) and 1 mM dithiothreitol (DTT) (AppliChem, Darmstadt, Germany) were added to each reaction tube, containing a single tissue slice.

    Techniques: Formalin-fixed Paraffin-Embedded, Sample Prep, Lysis, Protein Concentration, Mass Spectrometry, Molecular Weight