Structured Review

Amresco hepes
Ni 2+ and <t>L-cys</t> binding to coptisine (Cop) measured using UV-visible spectroscopy. 16 μM Cop in the absence or presence of different molar equivalents of Ni 2+ (1 mM NiCl 2 ) (A) or L-cys (1 mM) (B) in 20 mM <t>HEPES</t> buffer, pH 7.5. A-F, 0.0 to 320 μM of Ni 2+ ; A-F, 0.0 to 200 μM of L-cys. The inset shows the titration curves for binding of Ni 2+ or L-cys to Cop at 355 nm. Wavelength (nm) is represented on the x -axis and absorption (Abs) is represented on the y -axis.
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Images

1) Product Images from "Coptisine-induced inhibition of Helicobacter pylori: elucidation of specific mechanisms by probing urease active site and its maturation process"

Article Title: Coptisine-induced inhibition of Helicobacter pylori: elucidation of specific mechanisms by probing urease active site and its maturation process

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

doi: 10.1080/14756366.2018.1501044

Ni 2+ and L-cys binding to coptisine (Cop) measured using UV-visible spectroscopy. 16 μM Cop in the absence or presence of different molar equivalents of Ni 2+ (1 mM NiCl 2 ) (A) or L-cys (1 mM) (B) in 20 mM HEPES buffer, pH 7.5. A-F, 0.0 to 320 μM of Ni 2+ ; A-F, 0.0 to 200 μM of L-cys. The inset shows the titration curves for binding of Ni 2+ or L-cys to Cop at 355 nm. Wavelength (nm) is represented on the x -axis and absorption (Abs) is represented on the y -axis.
Figure Legend Snippet: Ni 2+ and L-cys binding to coptisine (Cop) measured using UV-visible spectroscopy. 16 μM Cop in the absence or presence of different molar equivalents of Ni 2+ (1 mM NiCl 2 ) (A) or L-cys (1 mM) (B) in 20 mM HEPES buffer, pH 7.5. A-F, 0.0 to 320 μM of Ni 2+ ; A-F, 0.0 to 200 μM of L-cys. The inset shows the titration curves for binding of Ni 2+ or L-cys to Cop at 355 nm. Wavelength (nm) is represented on the x -axis and absorption (Abs) is represented on the y -axis.

Techniques Used: Binding Assay, Spectroscopy, Titration

2) Product Images from "Coptisine-induced inhibition of Helicobacter pylori: elucidation of specific mechanisms by probing urease active site and its maturation process"

Article Title: Coptisine-induced inhibition of Helicobacter pylori: elucidation of specific mechanisms by probing urease active site and its maturation process

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

doi: 10.1080/14756366.2018.1501044

Ni 2+ and L-cys binding to coptisine (Cop) measured using UV-visible spectroscopy. 16 μM Cop in the absence or presence of different molar equivalents of Ni 2+ (1 mM NiCl 2 ) (A) or L-cys (1 mM) (B) in 20 mM HEPES buffer, pH 7.5. A-F, 0.0 to 320 μM of Ni 2+ ; A-F, 0.0 to 200 μM of L-cys. The inset shows the titration curves for binding of Ni 2+ or L-cys to Cop at 355 nm. Wavelength (nm) is represented on the x -axis and absorption (Abs) is represented on the y -axis.
Figure Legend Snippet: Ni 2+ and L-cys binding to coptisine (Cop) measured using UV-visible spectroscopy. 16 μM Cop in the absence or presence of different molar equivalents of Ni 2+ (1 mM NiCl 2 ) (A) or L-cys (1 mM) (B) in 20 mM HEPES buffer, pH 7.5. A-F, 0.0 to 320 μM of Ni 2+ ; A-F, 0.0 to 200 μM of L-cys. The inset shows the titration curves for binding of Ni 2+ or L-cys to Cop at 355 nm. Wavelength (nm) is represented on the x -axis and absorption (Abs) is represented on the y -axis.

Techniques Used: Binding Assay, Spectroscopy, Titration

Related Articles

Centrifugation:

Article Title: Human histone deacetylase 6 shows strong preference for tubulin dimers over assembled microtubules
Article Snippet: The lysate was then cleared by centrifugation at 40,000 × g for 30 min at 4 °C and the supernatant was loaded onto a Strep-Tactin column (IBA, Gottingen, Germany) equilibrated in the lysis buffer. .. The HDAC6 protein was then separated from tags and the TEV protease by size exclusion chromatography using the Superdex 16/600 HR200 column (GE Healthcare Bio-Sciences, Little Chalfont, UK) with 30 mM HEPES, 140 mM NaCl, 10 mM KCl, 3% glycerol, and 0.25 mM TCEP (AMRESCO, Solon, OH, USA), with a pH 7.4 adjusted with NaOH, as a mobile phase.

Stable Transfection:

Article Title: The Impact and Mechanism of a Novel Allosteric AMPA Receptor Modulator LCX001 on Protection Against Respiratory Depression in Rodents
Article Snippet: Whole-Cell Patch-Clamp Recording HEK293 cells that stably expressed AMPA-GluA2 receptors were seeded into the recording chamber 2 days before patch-clamp recordings, and the experiments were conducted in whole-cell configuration. .. The patch pipettes had open tips with resistance of 4–8 MΩ and were filled with internal electrode solution (mM): 135 CsCl (Sigma, Beijing, China), 10 CsF (Tocris, Bristol, United Kingdom), 10 HEPES (Amresco, United States), 5 Cs4BAPTA (Tocris, Bristol, United Kingdom), 1 MgCl2 , 0.5 CaCl2 , pH 7.2.

Cell Isolation:

Article Title: Genome-wide search for the genes accountable for the induced resistance to HIV-1 infection in activated CD4+ T cells: apparent transcriptional signatures, co-expression networks and possible cellular processes
Article Snippet: Resting CD4 + T cells were then purified by magnetic negative selection (CD4 + T cell Isolation Kit II, Miltenyi Biotec) and only the aliquots of cells with purity > 95% as determined by flow cytometry were used for further analysis. .. Briefly, freshly isolated CD4 + T cells were resuspended in RPMI 1640 medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Gibco) and 20 mM HEPES (Amresco) and seeded in 6-well plates either at an initial density of 2 × 106 cells/well with human recombinant IL-2 (100 U/ml) and PHA (5 μg/ml) or 0.5 × 106 cells/well with polystyrene beads coated anti-CD3/CD28 antibodies (Dynal beads CD3/CD28 T Cell Expander, Dynal) at a bead to cell ratio of 3:1.

Incubation:

Article Title: PGC-1α Promotes Ureagenesis in Mouse Periportal Hepatocytes through SIRT3 and SIRT5 in Response to Glucagon
Article Snippet: Periportal hepatocytes culture and treatment For the starvation experiments the medium was replaced by HBSS supplemented with 10 mM HEPES and an antibiotic solution (Amresco) as previously described . .. Periportal hepatocytes transfected with corresponding siRNAs were incubated with 4 mM L-alanine at 37 °C for 4 h in the presence or absence (control) of 1 μM glucagon.

Article Title: Synergistic antitumor activity of gemcitabine combined with triptolide in pancreatic cancer cells
Article Snippet: .. The cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), penicillin/streptomycin (100 U/ml each; Shijiazhuang No. 4 Pharmaceutical Co., Ltd., Shijiazhuang, China), sodium bicarbonate (2 g/l; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and HEPES (2.4 g/l; Amresco, LLC, Solon, OH, USA), and were incubated at 37°C with 5% CO2 . .. In vitro cytotoxicities of triptolide and gemcitabine, alone or in combination, in the pancreatic cancer cell lines were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay ( ).

Article Title: Human histone deacetylase 6 shows strong preference for tubulin dimers over assembled microtubules
Article Snippet: To further assist cell lysis, Igepal-630 was added to the cell lysate to a final concentration of 0.2% (v/v) and the mixture was incubated for 20 min on ice. .. The HDAC6 protein was then separated from tags and the TEV protease by size exclusion chromatography using the Superdex 16/600 HR200 column (GE Healthcare Bio-Sciences, Little Chalfont, UK) with 30 mM HEPES, 140 mM NaCl, 10 mM KCl, 3% glycerol, and 0.25 mM TCEP (AMRESCO, Solon, OH, USA), with a pH 7.4 adjusted with NaOH, as a mobile phase.

Article Title: Cryo-EM structure of the polycystic kidney disease-like channel PKD2L1
Article Snippet: Purification of truncated mutant proteins For cell harvesting, transfected cells were centrifuged at 800 × g and resuspended in lysis buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM EDTA and protease inhibitor cocktail (Amresco; 2 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml pepstanin). .. Then 2% dodecyl maltoside (DDM), 0.5% soybean lipids (Sigma), 0.4% cholesterol hemisuccinate​ (CHS) (Anatrace) was supplemented and the mixture was incubated for 1.5–2 h at 4 °C.

Activity Assay:

Article Title: Coptisine-induced inhibition of Helicobacter pylori: elucidation of specific mechanisms by probing urease active site and its maturation process
Article Snippet: .. Acetohydroxamic acid (AHA; C2 H5 NO2 , purity: 98%), jack bean urease (JBU; type III with activity of 31.66 U/mg solid), urea, dithiothreitol (DTT), L-cysteine (L-cys), glutathione (GSH), sodium fluoride (NaF), and HEPES (Amresco > 99%) were purchased from Sigma Aldrich (Steineheim, Germany). .. Boric acid (BA) was purchased from Aladdin Chemistry Company.

Infection:

Article Title: The Mitochondrial Na+/Ca2+ Exchanger Upregulates Glucose Dependent Ca2+ Signalling Linked to Insulin Secretion
Article Snippet: Ca2+ imaging was performed in pancreatic primary β cells and MIN6 cells attached onto coverslips and superfused with Ringer solution containing (in mM): 126 NaCl (Frutarom, 2355534700067), 5.4 KCl (Sigma, P9333), 0.8 MgCl2 (Gerbu, 1722), 20 HEPES (Amresco, 0511), 1.8 CaCl2 (Sigma, C1016), 15 Glucose (Gerbu, 2028), pH adjusted to 7.4 with NaOH (Sigma, S8045) or with high K+ (50 mM) Ringer solution when indicated. .. Mitochondrial Ca2+ measurements were performed in pancreatic primary β cells infected with lenti-pericam and in MIN6 cells expressing ratiometric mitochondrial pericam, which is targeted solely to the inner membrane of mitochondria , .

Expressing:

Article Title: The Mitochondrial Na+/Ca2+ Exchanger Upregulates Glucose Dependent Ca2+ Signalling Linked to Insulin Secretion
Article Snippet: Ca2+ imaging was performed in pancreatic primary β cells and MIN6 cells attached onto coverslips and superfused with Ringer solution containing (in mM): 126 NaCl (Frutarom, 2355534700067), 5.4 KCl (Sigma, P9333), 0.8 MgCl2 (Gerbu, 1722), 20 HEPES (Amresco, 0511), 1.8 CaCl2 (Sigma, C1016), 15 Glucose (Gerbu, 2028), pH adjusted to 7.4 with NaOH (Sigma, S8045) or with high K+ (50 mM) Ringer solution when indicated. .. Mitochondrial Ca2+ measurements were performed in pancreatic primary β cells infected with lenti-pericam and in MIN6 cells expressing ratiometric mitochondrial pericam, which is targeted solely to the inner membrane of mitochondria , .

Modification:

Article Title: Synergistic antitumor activity of gemcitabine combined with triptolide in pancreatic cancer cells
Article Snippet: .. The cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), penicillin/streptomycin (100 U/ml each; Shijiazhuang No. 4 Pharmaceutical Co., Ltd., Shijiazhuang, China), sodium bicarbonate (2 g/l; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and HEPES (2.4 g/l; Amresco, LLC, Solon, OH, USA), and were incubated at 37°C with 5% CO2 . .. In vitro cytotoxicities of triptolide and gemcitabine, alone or in combination, in the pancreatic cancer cell lines were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay ( ).

Article Title: Inhibition of IL-8-mediated endothelial adhesion, VSMCs proliferation and migration by siRNA-TMEM98 suggests TMEM98's emerging role in atherosclerosis
Article Snippet: Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Langley, OK, USA) supplemented with 10% fetal bovine serum (Gibco, Langley, OK, USA) at 37°C in a humidified atmosphere with 5% CO2 (CO2 incubator). .. The human umbilical vein endothelial cells (HUVECs) line was cultured in DMEM supplemented with 10% FBS, hepes (3.4 g/L) (Amresco, USA) at 37°C in a humidified atmosphere with 5% CO2 .

Derivative Assay:

Article Title: Inhibition of IL-8-mediated endothelial adhesion, VSMCs proliferation and migration by siRNA-TMEM98 suggests TMEM98's emerging role in atherosclerosis
Article Snippet: Cell culture The A7r5 cell line, originally derived from an embryonic rat aorta, was purchased from American Type Culture Collection (Manassas, VA, USA). .. The human umbilical vein endothelial cells (HUVECs) line was cultured in DMEM supplemented with 10% FBS, hepes (3.4 g/L) (Amresco, USA) at 37°C in a humidified atmosphere with 5% CO2 .

Article Title: Genome-wide search for the genes accountable for the induced resistance to HIV-1 infection in activated CD4+ T cells: apparent transcriptional signatures, co-expression networks and possible cellular processes
Article Snippet: Briefly, freshly isolated CD4 + T cells were resuspended in RPMI 1640 medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Gibco) and 20 mM HEPES (Amresco) and seeded in 6-well plates either at an initial density of 2 × 106 cells/well with human recombinant IL-2 (100 U/ml) and PHA (5 μg/ml) or 0.5 × 106 cells/well with polystyrene beads coated anti-CD3/CD28 antibodies (Dynal beads CD3/CD28 T Cell Expander, Dynal) at a bead to cell ratio of 3:1. .. Cells and the derived RNA samples and data sets were labeled “P”, “R”, and “B”, respectively, according to the PHA/IL-2 stimulated, un-stimulated resting and beads-stimulated settings.

Flow Cytometry:

Article Title: Genome-wide search for the genes accountable for the induced resistance to HIV-1 infection in activated CD4+ T cells: apparent transcriptional signatures, co-expression networks and possible cellular processes
Article Snippet: Resting CD4 + T cells were then purified by magnetic negative selection (CD4 + T cell Isolation Kit II, Miltenyi Biotec) and only the aliquots of cells with purity > 95% as determined by flow cytometry were used for further analysis. .. Briefly, freshly isolated CD4 + T cells were resuspended in RPMI 1640 medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Gibco) and 20 mM HEPES (Amresco) and seeded in 6-well plates either at an initial density of 2 × 106 cells/well with human recombinant IL-2 (100 U/ml) and PHA (5 μg/ml) or 0.5 × 106 cells/well with polystyrene beads coated anti-CD3/CD28 antibodies (Dynal beads CD3/CD28 T Cell Expander, Dynal) at a bead to cell ratio of 3:1.

Transfection:

Article Title: PGC-1α Promotes Ureagenesis in Mouse Periportal Hepatocytes through SIRT3 and SIRT5 in Response to Glucagon
Article Snippet: Periportal hepatocytes culture and treatment For the starvation experiments the medium was replaced by HBSS supplemented with 10 mM HEPES and an antibiotic solution (Amresco) as previously described . .. Periportal hepatocytes transfected with corresponding siRNAs were incubated with 4 mM L-alanine at 37 °C for 4 h in the presence or absence (control) of 1 μM glucagon.

Article Title: Cryo-EM structure of the polycystic kidney disease-like channel PKD2L1
Article Snippet: .. Purification of truncated mutant proteins For cell harvesting, transfected cells were centrifuged at 800 × g and resuspended in lysis buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM EDTA and protease inhibitor cocktail (Amresco; 2 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml pepstanin). ..

Cell Harvesting:

Article Title: Cryo-EM structure of the polycystic kidney disease-like channel PKD2L1
Article Snippet: .. Purification of truncated mutant proteins For cell harvesting, transfected cells were centrifuged at 800 × g and resuspended in lysis buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM EDTA and protease inhibitor cocktail (Amresco; 2 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml pepstanin). ..

Protease Inhibitor:

Article Title: Cryo-EM structure of the polycystic kidney disease-like channel PKD2L1
Article Snippet: .. Purification of truncated mutant proteins For cell harvesting, transfected cells were centrifuged at 800 × g and resuspended in lysis buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM EDTA and protease inhibitor cocktail (Amresco; 2 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml pepstanin). ..

Cell Culture:

Article Title: Molecular genetic characterization of p53 mutated oropharyngeal squamous cell carcinoma cells transformed with human papillomavirus E6 and E7 oncogenes
Article Snippet: .. The cell lines were cultured in RPMI-1640 (Welgene, Seoul, Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA), 1% penicillin/streptomycin (Gibco BRL), 10 mmol/l HEPES (Amresco Inc., Solon, OH, USA) at 37°C in a humidified incubator with 5% CO2 . .. Transfection with HPV16 E6 and E7 oncogenes We used both the HPV16 E6 and E7 oncogenes coding regions based on the sequences available from the HPV type 16 complete genome (GenBank: K02718.1).

Article Title: Synergistic antitumor activity of gemcitabine combined with triptolide in pancreatic cancer cells
Article Snippet: Paragraph title: Cell culture ... The cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), penicillin/streptomycin (100 U/ml each; Shijiazhuang No. 4 Pharmaceutical Co., Ltd., Shijiazhuang, China), sodium bicarbonate (2 g/l; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and HEPES (2.4 g/l; Amresco, LLC, Solon, OH, USA), and were incubated at 37°C with 5% CO2 .

Article Title: Inhibition of IL-8-mediated endothelial adhesion, VSMCs proliferation and migration by siRNA-TMEM98 suggests TMEM98's emerging role in atherosclerosis
Article Snippet: .. The human umbilical vein endothelial cells (HUVECs) line was cultured in DMEM supplemented with 10% FBS, hepes (3.4 g/L) (Amresco, USA) at 37°C in a humidified atmosphere with 5% CO2 . .. The THP-1 cell line was cultured in RPMI 1640 medium containing 10% FBS at 37°C in a humidified atmosphere with 5% CO2 .

other:

Article Title: Acidosis Promotes Bcl-2 Family-mediated Evasion of Apoptosis
Article Snippet: HEPES was purchased from Amresco (Solon, OH).

Article Title: Transcriptomic Characterization of Temperature Stress Responses in Larval Zebrafish
Article Snippet: Chemicals Most of chemicals with the highest purity available were obtained from China National Medicines Corporation LTD. Trypsin, BSA and HEPES were purchased from Amresco.

Article Title: Ca2+ is a Regulator of the WNK/OSR1/NKCC Pathway in a Human Salivary Gland Cell Line
Article Snippet: KCl, EGTA, Glucose, and HEPES was from Amresco (Solon, OH, USA).

Imaging:

Article Title: The Mitochondrial Na+/Ca2+ Exchanger Upregulates Glucose Dependent Ca2+ Signalling Linked to Insulin Secretion
Article Snippet: .. Ca2+ imaging was performed in pancreatic primary β cells and MIN6 cells attached onto coverslips and superfused with Ringer solution containing (in mM): 126 NaCl (Frutarom, 2355534700067), 5.4 KCl (Sigma, P9333), 0.8 MgCl2 (Gerbu, 1722), 20 HEPES (Amresco, 0511), 1.8 CaCl2 (Sigma, C1016), 15 Glucose (Gerbu, 2028), pH adjusted to 7.4 with NaOH (Sigma, S8045) or with high K+ (50 mM) Ringer solution when indicated. ..

Inverted Microscopy:

Article Title: The Mitochondrial Na+/Ca2+ Exchanger Upregulates Glucose Dependent Ca2+ Signalling Linked to Insulin Secretion
Article Snippet: Fluorescent Ca2+ imaging The imaging system consisted of an Axiovert 100 inverted microscope (Zeiss), Polychrome V monochromator (TILL Photonics, Planegg, Germany) and a SensiCam cooled charge-coupled device (PCO, Kelheim, Germany). .. Ca2+ imaging was performed in pancreatic primary β cells and MIN6 cells attached onto coverslips and superfused with Ringer solution containing (in mM): 126 NaCl (Frutarom, 2355534700067), 5.4 KCl (Sigma, P9333), 0.8 MgCl2 (Gerbu, 1722), 20 HEPES (Amresco, 0511), 1.8 CaCl2 (Sigma, C1016), 15 Glucose (Gerbu, 2028), pH adjusted to 7.4 with NaOH (Sigma, S8045) or with high K+ (50 mM) Ringer solution when indicated.

Recombinant:

Article Title: Genome-wide search for the genes accountable for the induced resistance to HIV-1 infection in activated CD4+ T cells: apparent transcriptional signatures, co-expression networks and possible cellular processes
Article Snippet: .. Briefly, freshly isolated CD4 + T cells were resuspended in RPMI 1640 medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Gibco) and 20 mM HEPES (Amresco) and seeded in 6-well plates either at an initial density of 2 × 106 cells/well with human recombinant IL-2 (100 U/ml) and PHA (5 μg/ml) or 0.5 × 106 cells/well with polystyrene beads coated anti-CD3/CD28 antibodies (Dynal beads CD3/CD28 T Cell Expander, Dynal) at a bead to cell ratio of 3:1. ..

Mutagenesis:

Article Title: Molecular genetic characterization of p53 mutated oropharyngeal squamous cell carcinoma cells transformed with human papillomavirus E6 and E7 oncogenes
Article Snippet: Both of the tongue cancer cell lines harbor p53 mutations, whereas the YD8 cell line has non-disruptive mutation that causes histidine to be substituted for by arginine at codon 273 in exon 8, the YD10B cell line has disruptive mutation that causes stop codon to be substitutes for tyrosine at codon 236 in exon 7 ( ). .. The cell lines were cultured in RPMI-1640 (Welgene, Seoul, Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA), 1% penicillin/streptomycin (Gibco BRL), 10 mmol/l HEPES (Amresco Inc., Solon, OH, USA) at 37°C in a humidified incubator with 5% CO2 .

Article Title: Cryo-EM structure of the polycystic kidney disease-like channel PKD2L1
Article Snippet: .. Purification of truncated mutant proteins For cell harvesting, transfected cells were centrifuged at 800 × g and resuspended in lysis buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM EDTA and protease inhibitor cocktail (Amresco; 2 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml pepstanin). ..

Isolation:

Article Title: Genome-wide search for the genes accountable for the induced resistance to HIV-1 infection in activated CD4+ T cells: apparent transcriptional signatures, co-expression networks and possible cellular processes
Article Snippet: .. Briefly, freshly isolated CD4 + T cells were resuspended in RPMI 1640 medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Gibco) and 20 mM HEPES (Amresco) and seeded in 6-well plates either at an initial density of 2 × 106 cells/well with human recombinant IL-2 (100 U/ml) and PHA (5 μg/ml) or 0.5 × 106 cells/well with polystyrene beads coated anti-CD3/CD28 antibodies (Dynal beads CD3/CD28 T Cell Expander, Dynal) at a bead to cell ratio of 3:1. ..

Size-exclusion Chromatography:

Article Title: Human histone deacetylase 6 shows strong preference for tubulin dimers over assembled microtubules
Article Snippet: .. The HDAC6 protein was then separated from tags and the TEV protease by size exclusion chromatography using the Superdex 16/600 HR200 column (GE Healthcare Bio-Sciences, Little Chalfont, UK) with 30 mM HEPES, 140 mM NaCl, 10 mM KCl, 3% glycerol, and 0.25 mM TCEP (AMRESCO, Solon, OH, USA), with a pH 7.4 adjusted with NaOH, as a mobile phase. .. Fluorometric assays Deacetylation activity of HDAC6 variants was determined using acetyl-Gly-Ala-(acetyl-Lys)-AMC (GAK(Ac) -AMC; Bachem, Bubendorf, Switzerland), Boc-(acetyl-Lys)-AMC (Boc-K(Ac)-AMC; Bachem) and Fluor-de-Lys (Enzo Life Sciences, Plymouth Meeting, PA, USA) substrates.

Labeling:

Article Title: Genome-wide search for the genes accountable for the induced resistance to HIV-1 infection in activated CD4+ T cells: apparent transcriptional signatures, co-expression networks and possible cellular processes
Article Snippet: Briefly, freshly isolated CD4 + T cells were resuspended in RPMI 1640 medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Gibco) and 20 mM HEPES (Amresco) and seeded in 6-well plates either at an initial density of 2 × 106 cells/well with human recombinant IL-2 (100 U/ml) and PHA (5 μg/ml) or 0.5 × 106 cells/well with polystyrene beads coated anti-CD3/CD28 antibodies (Dynal beads CD3/CD28 T Cell Expander, Dynal) at a bead to cell ratio of 3:1. .. Cells and the derived RNA samples and data sets were labeled “P”, “R”, and “B”, respectively, according to the PHA/IL-2 stimulated, un-stimulated resting and beads-stimulated settings.

Purification:

Article Title: Human histone deacetylase 6 shows strong preference for tubulin dimers over assembled microtubules
Article Snippet: Paragraph title: Purification of HDAC6 variants ... The HDAC6 protein was then separated from tags and the TEV protease by size exclusion chromatography using the Superdex 16/600 HR200 column (GE Healthcare Bio-Sciences, Little Chalfont, UK) with 30 mM HEPES, 140 mM NaCl, 10 mM KCl, 3% glycerol, and 0.25 mM TCEP (AMRESCO, Solon, OH, USA), with a pH 7.4 adjusted with NaOH, as a mobile phase.

Article Title: Genome-wide search for the genes accountable for the induced resistance to HIV-1 infection in activated CD4+ T cells: apparent transcriptional signatures, co-expression networks and possible cellular processes
Article Snippet: Resting CD4 + T cells were then purified by magnetic negative selection (CD4 + T cell Isolation Kit II, Miltenyi Biotec) and only the aliquots of cells with purity > 95% as determined by flow cytometry were used for further analysis. .. Briefly, freshly isolated CD4 + T cells were resuspended in RPMI 1640 medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Gibco) and 20 mM HEPES (Amresco) and seeded in 6-well plates either at an initial density of 2 × 106 cells/well with human recombinant IL-2 (100 U/ml) and PHA (5 μg/ml) or 0.5 × 106 cells/well with polystyrene beads coated anti-CD3/CD28 antibodies (Dynal beads CD3/CD28 T Cell Expander, Dynal) at a bead to cell ratio of 3:1.

Article Title: Cryo-EM structure of the polycystic kidney disease-like channel PKD2L1
Article Snippet: .. Purification of truncated mutant proteins For cell harvesting, transfected cells were centrifuged at 800 × g and resuspended in lysis buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM EDTA and protease inhibitor cocktail (Amresco; 2 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml pepstanin). ..

Cytometry:

Article Title: Genome-wide search for the genes accountable for the induced resistance to HIV-1 infection in activated CD4+ T cells: apparent transcriptional signatures, co-expression networks and possible cellular processes
Article Snippet: Resting CD4 + T cells were then purified by magnetic negative selection (CD4 + T cell Isolation Kit II, Miltenyi Biotec) and only the aliquots of cells with purity > 95% as determined by flow cytometry were used for further analysis. .. Briefly, freshly isolated CD4 + T cells were resuspended in RPMI 1640 medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Gibco) and 20 mM HEPES (Amresco) and seeded in 6-well plates either at an initial density of 2 × 106 cells/well with human recombinant IL-2 (100 U/ml) and PHA (5 μg/ml) or 0.5 × 106 cells/well with polystyrene beads coated anti-CD3/CD28 antibodies (Dynal beads CD3/CD28 T Cell Expander, Dynal) at a bead to cell ratio of 3:1.

Software:

Article Title: The Impact and Mechanism of a Novel Allosteric AMPA Receptor Modulator LCX001 on Protection Against Respiratory Depression in Rodents
Article Snippet: The patch pipettes had open tips with resistance of 4–8 MΩ and were filled with internal electrode solution (mM): 135 CsCl (Sigma, Beijing, China), 10 CsF (Tocris, Bristol, United Kingdom), 10 HEPES (Amresco, United States), 5 Cs4BAPTA (Tocris, Bristol, United Kingdom), 1 MgCl2 , 0.5 CaCl2 , pH 7.2. .. The holding potential was set as -80 mV by an Axopatch-700B amplifier (Axon Instruments, CA, United States), and pClamp8 software (Axon Instruments, CA, United States) was used for data acquisition.

Selection:

Article Title: Genome-wide search for the genes accountable for the induced resistance to HIV-1 infection in activated CD4+ T cells: apparent transcriptional signatures, co-expression networks and possible cellular processes
Article Snippet: Resting CD4 + T cells were then purified by magnetic negative selection (CD4 + T cell Isolation Kit II, Miltenyi Biotec) and only the aliquots of cells with purity > 95% as determined by flow cytometry were used for further analysis. .. Briefly, freshly isolated CD4 + T cells were resuspended in RPMI 1640 medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Gibco) and 20 mM HEPES (Amresco) and seeded in 6-well plates either at an initial density of 2 × 106 cells/well with human recombinant IL-2 (100 U/ml) and PHA (5 μg/ml) or 0.5 × 106 cells/well with polystyrene beads coated anti-CD3/CD28 antibodies (Dynal beads CD3/CD28 T Cell Expander, Dynal) at a bead to cell ratio of 3:1.

Patch Clamp:

Article Title: The Impact and Mechanism of a Novel Allosteric AMPA Receptor Modulator LCX001 on Protection Against Respiratory Depression in Rodents
Article Snippet: Paragraph title: Whole-Cell Patch-Clamp Recording ... The patch pipettes had open tips with resistance of 4–8 MΩ and were filled with internal electrode solution (mM): 135 CsCl (Sigma, Beijing, China), 10 CsF (Tocris, Bristol, United Kingdom), 10 HEPES (Amresco, United States), 5 Cs4BAPTA (Tocris, Bristol, United Kingdom), 1 MgCl2 , 0.5 CaCl2 , pH 7.2.

Concentration Assay:

Article Title: Effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca2+]i in the cells
Article Snippet: Chemical reagents: RPMI 1640 culture medium was supplied from HyClone Laboratories; fetal bovine serum from Hangzhou Sijiqing Biological Engineering Materials Co.,Ltd; pancreatin from Gibio; ethidium bromide (EB) from AMRESCO; acridine orange (AO) from AMRESCO; Fluo-3/AM from Biotium, US; TMRE(molecular probe) from Biotium, US; HEPES from AMRESCO. .. Preparation of the reagents: Fluo-3/AM 0.1 mg/stick was diluted to suitable concentration with DMSO, and then separately loaded and stored in the dark.

Article Title: Human histone deacetylase 6 shows strong preference for tubulin dimers over assembled microtubules
Article Snippet: NaCl was added to a final concentration of 150 mM. .. The HDAC6 protein was then separated from tags and the TEV protease by size exclusion chromatography using the Superdex 16/600 HR200 column (GE Healthcare Bio-Sciences, Little Chalfont, UK) with 30 mM HEPES, 140 mM NaCl, 10 mM KCl, 3% glycerol, and 0.25 mM TCEP (AMRESCO, Solon, OH, USA), with a pH 7.4 adjusted with NaOH, as a mobile phase.

Lysis:

Article Title: Human histone deacetylase 6 shows strong preference for tubulin dimers over assembled microtubules
Article Snippet: Following the washing step with the lysis buffer, the fusion protein was eluted using 50 mM Tris-HCl, 150 mM NaCl, 10 mM KCl, 10% glycerol and 3 mM desthiobiotin (pH 8.0). .. The HDAC6 protein was then separated from tags and the TEV protease by size exclusion chromatography using the Superdex 16/600 HR200 column (GE Healthcare Bio-Sciences, Little Chalfont, UK) with 30 mM HEPES, 140 mM NaCl, 10 mM KCl, 3% glycerol, and 0.25 mM TCEP (AMRESCO, Solon, OH, USA), with a pH 7.4 adjusted with NaOH, as a mobile phase.

Article Title: Cryo-EM structure of the polycystic kidney disease-like channel PKD2L1
Article Snippet: .. Purification of truncated mutant proteins For cell harvesting, transfected cells were centrifuged at 800 × g and resuspended in lysis buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM EDTA and protease inhibitor cocktail (Amresco; 2 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml pepstanin). ..

Gradient Centrifugation:

Article Title: Genome-wide search for the genes accountable for the induced resistance to HIV-1 infection in activated CD4+ T cells: apparent transcriptional signatures, co-expression networks and possible cellular processes
Article Snippet: Isolation and stimulation of CD4+ T cells Buffy coats were obtained from healthy donors (Kunming Blood Station) and peripheral blood mononuclear cells (PBMCs) were isolated by Ficool-Hypaque (TBD Sciences) gradient centrifugation. .. Briefly, freshly isolated CD4 + T cells were resuspended in RPMI 1640 medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Gibco) and 20 mM HEPES (Amresco) and seeded in 6-well plates either at an initial density of 2 × 106 cells/well with human recombinant IL-2 (100 U/ml) and PHA (5 μg/ml) or 0.5 × 106 cells/well with polystyrene beads coated anti-CD3/CD28 antibodies (Dynal beads CD3/CD28 T Cell Expander, Dynal) at a bead to cell ratio of 3:1.

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  • 99
    Amresco hepes
    Ni 2+ and <t>L-cys</t> binding to coptisine (Cop) measured using UV-visible spectroscopy. 16 μM Cop in the absence or presence of different molar equivalents of Ni 2+ (1 mM NiCl 2 ) (A) or L-cys (1 mM) (B) in 20 mM <t>HEPES</t> buffer, pH 7.5. A-F, 0.0 to 320 μM of Ni 2+ ; A-F, 0.0 to 200 μM of L-cys. The inset shows the titration curves for binding of Ni 2+ or L-cys to Cop at 355 nm. Wavelength (nm) is represented on the x -axis and absorption (Abs) is represented on the y -axis.
    Hepes, supplied by Amresco, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ni 2+ and L-cys binding to coptisine (Cop) measured using UV-visible spectroscopy. 16 μM Cop in the absence or presence of different molar equivalents of Ni 2+ (1 mM NiCl 2 ) (A) or L-cys (1 mM) (B) in 20 mM HEPES buffer, pH 7.5. A-F, 0.0 to 320 μM of Ni 2+ ; A-F, 0.0 to 200 μM of L-cys. The inset shows the titration curves for binding of Ni 2+ or L-cys to Cop at 355 nm. Wavelength (nm) is represented on the x -axis and absorption (Abs) is represented on the y -axis.

    Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

    Article Title: Coptisine-induced inhibition of Helicobacter pylori: elucidation of specific mechanisms by probing urease active site and its maturation process

    doi: 10.1080/14756366.2018.1501044

    Figure Lengend Snippet: Ni 2+ and L-cys binding to coptisine (Cop) measured using UV-visible spectroscopy. 16 μM Cop in the absence or presence of different molar equivalents of Ni 2+ (1 mM NiCl 2 ) (A) or L-cys (1 mM) (B) in 20 mM HEPES buffer, pH 7.5. A-F, 0.0 to 320 μM of Ni 2+ ; A-F, 0.0 to 200 μM of L-cys. The inset shows the titration curves for binding of Ni 2+ or L-cys to Cop at 355 nm. Wavelength (nm) is represented on the x -axis and absorption (Abs) is represented on the y -axis.

    Article Snippet: Acetohydroxamic acid (AHA; C2 H5 NO2 , purity: 98%), jack bean urease (JBU; type III with activity of 31.66 U/mg solid), urea, dithiothreitol (DTT), L-cysteine (L-cys), glutathione (GSH), sodium fluoride (NaF), and HEPES (Amresco > 99%) were purchased from Sigma Aldrich (Steineheim, Germany).

    Techniques: Binding Assay, Spectroscopy, Titration

    Ni 2+ and L-cys binding to coptisine (Cop) measured using UV-visible spectroscopy. 16 μM Cop in the absence or presence of different molar equivalents of Ni 2+ (1 mM NiCl 2 ) (A) or L-cys (1 mM) (B) in 20 mM HEPES buffer, pH 7.5. A-F, 0.0 to 320 μM of Ni 2+ ; A-F, 0.0 to 200 μM of L-cys. The inset shows the titration curves for binding of Ni 2+ or L-cys to Cop at 355 nm. Wavelength (nm) is represented on the x -axis and absorption (Abs) is represented on the y -axis.

    Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

    Article Title: Coptisine-induced inhibition of Helicobacter pylori: elucidation of specific mechanisms by probing urease active site and its maturation process

    doi: 10.1080/14756366.2018.1501044

    Figure Lengend Snippet: Ni 2+ and L-cys binding to coptisine (Cop) measured using UV-visible spectroscopy. 16 μM Cop in the absence or presence of different molar equivalents of Ni 2+ (1 mM NiCl 2 ) (A) or L-cys (1 mM) (B) in 20 mM HEPES buffer, pH 7.5. A-F, 0.0 to 320 μM of Ni 2+ ; A-F, 0.0 to 200 μM of L-cys. The inset shows the titration curves for binding of Ni 2+ or L-cys to Cop at 355 nm. Wavelength (nm) is represented on the x -axis and absorption (Abs) is represented on the y -axis.

    Article Snippet: Acetohydroxamic acid (AHA; C2 H5 NO2 , purity: 98%), jack bean urease (JBU; type III with activity of 31.66 U/mg solid), urea, dithiothreitol (DTT), L-cysteine (L-cys), glutathione (GSH), sodium fluoride (NaF), and HEPES (Amresco > 99%) were purchased from Sigma Aldrich (Steineheim, Germany).

    Techniques: Binding Assay, Spectroscopy, Titration