ethylenediaminetetraacetic acid tetrasodium salt hydrate  (Millipore)


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    Name:
    Ethylenediaminetetraacetic acid tetrasodium salt hydrate
    Description:

    Catalog Number:
    ed4s
    Price:
    None
    Applications:
    EDTA is an inhibitor of metalloproteases, at effective concentrations of 1-10 mM. EDTA acts as a chelator of the zinc ion in the active site of metalloproteases, and can also inhibit other metal ion-dependent proteases such as calcium-dependent cysteine proteases. For use as an anticoagulant, disodium or tripotassium salts of EDTA are most commonly used. The optimal concentration is 1.5 mg per ml of blood.
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    Structured Review

    Millipore ethylenediaminetetraacetic acid tetrasodium salt hydrate
    Ethylenediaminetetraacetic acid tetrasodium salt hydrate

    https://www.bioz.com/result/ethylenediaminetetraacetic acid tetrasodium salt hydrate/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ethylenediaminetetraacetic acid tetrasodium salt hydrate - by Bioz Stars, 2020-03
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    Related Products / Commonly Used Together

    tissue permeability
    ihc staining

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    Related Articles

    Mass Spectrometry:

    Article Title: LARGE SCALE PURIFICATION OF BUTYRYLCHOLINESTERASE FROM HUMAN PLASMA SUITABLE FOR INJECTION INTO MONKEYS; A POTENTIAL NEW THERAPEUTIC FOR PROTECTION AGAINST COCAINE AND NERVE AGENT TOXICITY
    Article Snippet: .. The 100x buffer was made in a glass bottle and contained 152 g EDTA tetrasodium salt (Aldrich E2,629-0, 98% pure) plus 456 ml glacial acetic acid, and 18 g sodium hydroxide in a total volume of 4 L. After 1:100 dilution this buffer had a conductivity of 0.36 mS and a pH of 4.0. ..

    Blocking Assay:

    Article Title: Vascular Associations and Dynamic Process Motility in Perivascular Myeloid Cells of the Mouse Choroid: Implications for Function and Senescent Change
    Article Snippet: For immunohistochemical analyses, choroidoscleral explants were washed with PBS for 1 hour, incubated in 20 mM of ethylenediaminetetraacetic acid (EDTA) tetrasodium salt hydrate at 37°C for 30 min, and permeabilized with a solution of PBS containing 0.5% Triton X-100 (PBST) for 1 hour (all reagents from Sigma-Aldrich, St. Louis, MO). .. Explants were transferred to a blocking solution (PBST containing 2% goat serum) for 30 minutes and incubated overnight with the following primary antibodies: anti-major histocompatibility complex class II (MHCII, 1:1000 dilution; BD Pharmingen, San Diego, CA), anti-ionized calcium binding adaptor molecule-1 (Iba1, 1:500 dilution; Wako, Richmond, VA), anti CD-163 (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and anti-CD169 (1:500 dilution; AbD Serotec, Raleigh, NC).

    Article Title: Comparative Anatomy of the Mammalian Corneal Subbasal Nerve Plexus
    Article Snippet: IHC Staining To enhance tissue permeability and optimize IHC staining, corneas from all animals except mice were first permeabilized in 0.01% hyaluronidase (type IV-S, catalogue #H4272, Sigma-Aldrich, Corp., St. Louis, MO, USA) and 0.1% ethylenediaminetetraacetic acid (EDTA) (catalogue #E5391, Sigma-Aldrich, Corp.) in 0.1 M PBS, pH 5.3. .. All tissues were then rinsed three times for 20 minutes each in PBS + 0.3% Triton X-100 (PBS-TX), incubated for 2 hours in blocking serum (1% bovine serum albumin in PBS-TX) and incubated for 4 days (macaques) or overnight (all other species) at RT on a rocker table in either a rabbit polyclonal antibody (mouse corneas) or a mouse monoclonal antibody (all other corneas) against neuronal class III β-tubulin (TuJ1, 1:500, Covance Research Products, Berkeley, CA, USA).

    Incubation:

    Article Title: Vascular Associations and Dynamic Process Motility in Perivascular Myeloid Cells of the Mouse Choroid: Implications for Function and Senescent Change
    Article Snippet: .. For immunohistochemical analyses, choroidoscleral explants were washed with PBS for 1 hour, incubated in 20 mM of ethylenediaminetetraacetic acid (EDTA) tetrasodium salt hydrate at 37°C for 30 min, and permeabilized with a solution of PBS containing 0.5% Triton X-100 (PBST) for 1 hour (all reagents from Sigma-Aldrich, St. Louis, MO). .. Explants were transferred to a blocking solution (PBST containing 2% goat serum) for 30 minutes and incubated overnight with the following primary antibodies: anti-major histocompatibility complex class II (MHCII, 1:1000 dilution; BD Pharmingen, San Diego, CA), anti-ionized calcium binding adaptor molecule-1 (Iba1, 1:500 dilution; Wako, Richmond, VA), anti CD-163 (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and anti-CD169 (1:500 dilution; AbD Serotec, Raleigh, NC).

    Article Title: A Quadrupole Dalton-based multi-attribute method for product characterization, process development, and quality control of therapeutic proteins
    Article Snippet: .. The alkylated and denatured antibodies were then diluted using 100 mM sodium phosphate pH 7.0 and 0.1 mM ethylenediaminetetraacetic acid (EDTA, E5391, Sigma) to reduce guanidine concentration to 1.7 M. Lysyl endopeptidase (Lys-C, 125–05061, Wako) was added at 1:5 protease: protein ratio and incubated at 37°C for 4 hours. .. After digestion, the samples were either stored at −80°C or transferred to HPLC vials and placed in a temperature controlled (6–8°C) auto sampler for analysis.

    Article Title: Comparative Anatomy of the Mammalian Corneal Subbasal Nerve Plexus
    Article Snippet: IHC Staining To enhance tissue permeability and optimize IHC staining, corneas from all animals except mice were first permeabilized in 0.01% hyaluronidase (type IV-S, catalogue #H4272, Sigma-Aldrich, Corp., St. Louis, MO, USA) and 0.1% ethylenediaminetetraacetic acid (EDTA) (catalogue #E5391, Sigma-Aldrich, Corp.) in 0.1 M PBS, pH 5.3. .. Rat and guinea pig corneas were incubated in the permeability reagent overnight at RT, whereas corneas from all larger mammals were incubated for 24 to 48 hours at 37°C.

    Expressing:

    Article Title: Reporter-Based Isolation of Developmental Myogenic Progenitors
    Article Snippet: WT and mutant mice expressing a reporter in myogenic progenitors (Table ). .. 1x phosphate buffer saline (PBS) pH 7.4 (ThermoFisher Scientific 10010031) Ethanol 70% (vol/vol) DMEM (High Glucose) (ThermoFisher Scientific 10938025) Penicillin/streptomycin mixtures (P/S) (100X, Omega Scientific PS-20) HEPES 1M (ThermoFisher Scientific 15630080) L-Glutamine 200 mM (ThermoFisher Scientific 25030081) 1x Ca2+ -free Hanks' balanced salt solution (HBSS) (ThermoFisher Scientific 14170070) Bovine serum albumin (BSA) (Sigma A1933) Glucose (Sigma G7021) Magnesium sulfate (MgSO4 ) (Sigma M2643) Sodium hydroxide (NaOH) 1N (Sigma S2770) EDTA disodium salt (Sigma E6635) Ammonium chloride (NH4 Cl) (Sigma A9434) Potassium bicarbonate (KHCO3) (Sigma 60339) EDTA tetrasodium salt hydrate (Sigma E5391) Fetal bovine serum (FBS) (ThermoFisher Scientific 10270106) Collagenase Type V (Sigma C9263) Dispase (ThermoFisher Scientific 17105041) DNAse I (Worthington ) Parafilm Horse serum (ThermoFisher Scientific 16050122) Calf skin collagen (Sigma C8919) ECM gel from Engelbreth-Holm-Swarm murine sarcoma (Sigma E1270) Ultrapure distilled water (ThermoFisher Scientific 10977023) 16% Formaldehyde (w/v), Methanol-free (ThermoFisher Scientific 28906) CAUTION Hazardous in case of eye contact and on ingestion Anti-Pax7 (Developmental Studies Hybridoma Bank AB_528428) Anti-MyoD (Dako M3512) Anti-Myogenin (BD Pharmingen 556358) Anti-Desmin (Sigma SAB5600054) DAPI (diamidino-2-phenylindole) (Sigma 32670) Ice Liquid nitrogen.

    Derivative Assay:

    Article Title: STABILITY OF HETEROGENEOUS ELASTOGRAPHY PHANTOMS MADE FROM OIL DISPERSIONS IN AQUEOUS GELS
    Article Snippet: First, make an aqueous gelatin solution by mixing together 307 mL of distilled water (at room temperature) with 54 g of (dry) 200 bloom gelatin derived from calfskin. (Vyse Gelatin Co., Schiller Park, IL, USA) After covering the beaker with a thin plastic membrane, such as Saran Wrap® , heat the mixture in a double boiler to about 90 °C until the mixture becomes optically transparent. .. Add 2.73 g of EDTA tetrasodium salt hydrate (catalog no. E26290, Aldrich Chemical Co., Inc., Milwaukee, WI, USA), 0.93 g CuCl2 -2H2 O, 6.4 g NaCl and 12 g of liquid Germall-plus® (Sutton Laboratories, Inc., Chatham, NJ, USA).

    High Performance Liquid Chromatography:

    Article Title: Antiaflatoxigenic effect of fullerene C60 nanoparticles at environmentally plausible concentrations
    Article Snippet: Superoxide dismutase from bovine erythrocytes (3000 U/mg protein) (SOD), glutathione reductase from baker’s yeast ( Saccharomyces cerevisiae ) (100–300 U/mg protein) (GR), xanthine oxidase from bovine milk (0.4–1.0 U/mg protein) (XOD), xanthine, nitrotetrazolium blue chloride (NBT), ethylenediaminetetraacetic acid tetrasodium salt (EDTA-4Na), l -glutathione, reduced (GSH), l -glutathione oxidised disodium salt (GSSG), potassium cyanide, diethylenetriaminepentaacetic acid (DTPA), N -ethylmaleimide (NEM) and sodium dithionite (DT) were purchased by Sigma Aldrich (Germany). β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt (NADPH) was purchased from Serva (Germany), while stabilized 3% solution of hydrogen peroxide (H2 O2 ), o -phthalaldehyde (OPA) and formic acid were purchased from Fluka (Germany). .. Acetonitrile and methanol (both HPLC grade) were obtained from J. T. Baker (Italy), while yeast extract, potato dextrose agar and sucrose were purchased from Biolife (Italy).

    Article Title: A Quadrupole Dalton-based multi-attribute method for product characterization, process development, and quality control of therapeutic proteins
    Article Snippet: The alkylated and denatured antibodies were then diluted using 100 mM sodium phosphate pH 7.0 and 0.1 mM ethylenediaminetetraacetic acid (EDTA, E5391, Sigma) to reduce guanidine concentration to 1.7 M. Lysyl endopeptidase (Lys-C, 125–05061, Wako) was added at 1:5 protease: protein ratio and incubated at 37°C for 4 hours. .. After digestion, the samples were either stored at −80°C or transferred to HPLC vials and placed in a temperature controlled (6–8°C) auto sampler for analysis.

    Article Title: ER Microsome Preparation and Subsequent IAA Quantification in Maize Coleoptile and Primary Root Tissue
    Article Snippet: .. Pipettes (1 ml, 200 µl and 10 µl) (Gilson Scientific Ltd., catalog number: F123602, F123601 and F144802) Eppendorf tubes (2 ml) (Eppendorf AG, catalog number: 0030120094) Eppendorf conical tubes (50 ml) (Eppendorf AG, catalog number: 0030122178) 0.45 µm syringe filters (Sigma-Aldrich, catalog number: F8273) Cheese cloth (DUTSCHER SCIENTIFIC, catalog number: 789056) Ice box and ice Liquid nitrogen Triethanolamine-acetic acid (TEA-HOAc) (Sigma-Aldrich, catalog number: 90278) Potassium acetate (KOAc) (Sigma-Aldrich, catalog number: P1190) Magnesium acetate [Mg(OAc)2 ] (Sigma-Aldrich, catalog number: M5661) Sucrose (Sigma-Aldrich, catalog number: S0389) DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815) Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E5391) Tris-Hydrochloride (Tris-HCl) (Sigma-Aldrich, catalog number: 252859) β-Nicotinamide adenine dinucleotide phosphate disodium salt (NADPH) (Sigma-Aldrich, catalog number: 93205) Flavin adenine dinucleotide (FAD) (Sigma-Aldrich, catalog number: F6625) Indole-3-pyruvic acid (IPyA) (Sigma-Aldrich, catalog number: I7017) Tryptophan (Sigma-Aldrich, catalog number: T0254) Ethyl acetate (Sigma-Aldrich, catalog number: 270989) Sodium carbonate (Na2CO3) (Sigma-Aldrich, catalog number: S7795) Distilled water Ethyl acetate for HPLC (Sigma-Aldrich, catalog number: 650528) Acetic acid for HPLC (Sigma-Aldrich, catalog number: A6283) Methanol for HPLC (Sigma-Aldrich, catalog number: 34860) Stock solutions (see ) Buffers (see ) Buffers for microsome extraction (Buffer 1, Buffer 2, Buffer 3 and Buffer 4) Buffers for HPLC analysis .. Porcelain mortar & pestle (Sigma-Aldrich, catalog number: Z247464 and Z247502) Glass bottles for buffers (100 ml, 250 ml and 500 ml) (Sigma-Aldrich, catalog number: Z305170, Z305189 and Z305197) Refrigerated table centrifuge (Eppendorf AG, model: 5430R) Ultracentrifuge with swing-out rotor SW41 (Beckman Coulter, catalog number: 331362) Ultracentrifuge corex tubes (Beckman Coulter, catalog number: 331372) Glass rod and a 2 ml Potter–Elvehjem homogenizer (Sigma-Aldrich, catalog number: P7734-1EA) water bath (37 °C) (Thermo Fisher Scientific, model: TSGP02) Speed-vac (LABCONCO, catalog number: 7810016) Nanodrop ND-2000 (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 13400504) High-performance liquid chromatography (HPLC) (600E multisolvent delivery system) (WATERS, catalog number: 720000699EN) Reverse phase column (Apollo C18) (250 mm x 4.6 mm, 5 μm) (Thermo Fisher Scientific, Grace™, catalog number: 5126767) UV monitor 486 (WATERS) and a fluorescence monitor 470 (WATERS)

    Immunohistochemistry:

    Article Title: Vascular Associations and Dynamic Process Motility in Perivascular Myeloid Cells of the Mouse Choroid: Implications for Function and Senescent Change
    Article Snippet: .. For immunohistochemical analyses, choroidoscleral explants were washed with PBS for 1 hour, incubated in 20 mM of ethylenediaminetetraacetic acid (EDTA) tetrasodium salt hydrate at 37°C for 30 min, and permeabilized with a solution of PBS containing 0.5% Triton X-100 (PBST) for 1 hour (all reagents from Sigma-Aldrich, St. Louis, MO). .. Explants were transferred to a blocking solution (PBST containing 2% goat serum) for 30 minutes and incubated overnight with the following primary antibodies: anti-major histocompatibility complex class II (MHCII, 1:1000 dilution; BD Pharmingen, San Diego, CA), anti-ionized calcium binding adaptor molecule-1 (Iba1, 1:500 dilution; Wako, Richmond, VA), anti CD-163 (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and anti-CD169 (1:500 dilution; AbD Serotec, Raleigh, NC).

    Article Title: Comparative Anatomy of the Mammalian Corneal Subbasal Nerve Plexus
    Article Snippet: .. IHC Staining To enhance tissue permeability and optimize IHC staining, corneas from all animals except mice were first permeabilized in 0.01% hyaluronidase (type IV-S, catalogue #H4272, Sigma-Aldrich, Corp., St. Louis, MO, USA) and 0.1% ethylenediaminetetraacetic acid (EDTA) (catalogue #E5391, Sigma-Aldrich, Corp.) in 0.1 M PBS, pH 5.3. .. Rat and guinea pig corneas were incubated in the permeability reagent overnight at RT, whereas corneas from all larger mammals were incubated for 24 to 48 hours at 37°C.

    Concentration Assay:

    Article Title: A Quadrupole Dalton-based multi-attribute method for product characterization, process development, and quality control of therapeutic proteins
    Article Snippet: .. The alkylated and denatured antibodies were then diluted using 100 mM sodium phosphate pH 7.0 and 0.1 mM ethylenediaminetetraacetic acid (EDTA, E5391, Sigma) to reduce guanidine concentration to 1.7 M. Lysyl endopeptidase (Lys-C, 125–05061, Wako) was added at 1:5 protease: protein ratio and incubated at 37°C for 4 hours. .. After digestion, the samples were either stored at −80°C or transferred to HPLC vials and placed in a temperature controlled (6–8°C) auto sampler for analysis.

    Cell Culture:

    Article Title: Inactivation of Retinoblastoma Protein (Rb1) in the Oocyte: Evidence That Dysregulated Follicle Growth Drives Ovarian Teratoma Formation in Mice
    Article Snippet: .. When no evidence of parthenogenesis was observed, we conducted a second series of experiments replicating a previously described culture protocol [ ] in which GV oocytes were collected and cultured in Minimal Essential Medium (MEM) with Earle’s balanced salt solution (Sigma, MO, USA) supplemented with essential amino acids (Sigma, MO, USA), Penicillin and Streptomycin (Gibco, Life Technologies, CA, USA), 0.01 mM Ethylenediaminetetraacetic acid tetrasodium salt hydrate (Tetrasodium EDTA, Sigma, USA), 0.23 mM pyruvic acid (Sigma, USA) and 3% bovine serum albumin (BSA, Sigma, USA). .. GV breakdown (GVBD), polar body (PB) extrusion and parthenogenetic activation were assessed as described previously [ ].

    other:

    Article Title: A quantitative PCR method for measuring absolute telomere length
    Article Snippet: Buccal cell buffer Prepare 1 litre of the buccal buffer, [0.01 M Tris-hydrochloride (Sigma T-3253), 0.1 M ethylenediaminetetraacetic acid tetra Na salt (Sigma E5391), 0.02 M sodium chloride (Sigma S5886)].

    Article Title: Effects of Cholesterol Incorporation on the Physicochemical, Colloidal, and Biological Characteristics of pH-sensitive AB2 Miktoarm Polymer-Based Polymersomes
    Article Snippet: Thionyl chloride, propargylamine, methyl acrylate, ethylenediamine, methoxy poly(ethylene glycol) (mPEG-OH, Mn 2 kDa), p -toluenesulfonyl chloride, ethylenediaminetetraacetic acid (EDTA) tetrasodium salt hydrate, copper (II) sulfate pentahydrate, sodium l -ascorbate, 2-mercaptoethanol, pyrene, RPMI1640 medium, 4-(2-hydroxy-ethyl)-1-piperazine (HEPES), l -glutamine, d -glucose, acetone, dimethylsulfoxide (DMSO), dimethylformamide (DMF), methanol, 1,4-dioxane, diethyl ether, phosphate buffered saline (PBS), 5(6)-carboxyfluorescein (CF), cholesterol (CL), paraformaldehyde (PFA), sodium tetraborate, boric acid, and Triton X-100 were purchased from Sigma-Aldrich Co. (St. Louis, MO).

    Article Title: Molecular interactions of plant oil components with stratum corneum lipids correlate with clinical measures of skin barrier function
    Article Snippet: Materials Bovine brain ceramide (type III), cholesterol, palmitic acid, oleic acid (99%), glyceryl trioleate (99%), deuterated oleic acid (OA-d34 , 98 atom% D), chloroform (99%), 1-octanol (99.5%), sodium chloride (99%) and EDTA tetrasodium salt (99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Polymerase Chain Reaction:

    Article Title: Rapid Detection of Deletion, Insertion, and Substitution Mutations via Heteroduplex Analysis Using Capillary- and Microchip-Based Electrophoresis
    Article Snippet: GeneAmp thin-walled PCR tubes, 10× PCR buffer, 25 mM MgCl2 , 100 mM dNTPs stock solutions, and Taq DNA polymerase (5 unit/μl) were from Perkin-Elmer. .. Boric acid, ethylenediaminetetraacetic acid tetrasodium salt (EDTA) and tris[hydroxymethyl]aminomethane (Tris) were from Sigma Chemical.

    Injection:

    Article Title: Inactivation of Retinoblastoma Protein (Rb1) in the Oocyte: Evidence That Dysregulated Follicle Growth Drives Ovarian Teratoma Formation in Mice
    Article Snippet: Germinal vesicle (GV) stage oocytes were collected 44–45 h after PMSG injection and cultured. .. When no evidence of parthenogenesis was observed, we conducted a second series of experiments replicating a previously described culture protocol [ ] in which GV oocytes were collected and cultured in Minimal Essential Medium (MEM) with Earle’s balanced salt solution (Sigma, MO, USA) supplemented with essential amino acids (Sigma, MO, USA), Penicillin and Streptomycin (Gibco, Life Technologies, CA, USA), 0.01 mM Ethylenediaminetetraacetic acid tetrasodium salt hydrate (Tetrasodium EDTA, Sigma, USA), 0.23 mM pyruvic acid (Sigma, USA) and 3% bovine serum albumin (BSA, Sigma, USA).

    Binding Assay:

    Article Title: Vascular Associations and Dynamic Process Motility in Perivascular Myeloid Cells of the Mouse Choroid: Implications for Function and Senescent Change
    Article Snippet: For immunohistochemical analyses, choroidoscleral explants were washed with PBS for 1 hour, incubated in 20 mM of ethylenediaminetetraacetic acid (EDTA) tetrasodium salt hydrate at 37°C for 30 min, and permeabilized with a solution of PBS containing 0.5% Triton X-100 (PBST) for 1 hour (all reagents from Sigma-Aldrich, St. Louis, MO). .. Explants were transferred to a blocking solution (PBST containing 2% goat serum) for 30 minutes and incubated overnight with the following primary antibodies: anti-major histocompatibility complex class II (MHCII, 1:1000 dilution; BD Pharmingen, San Diego, CA), anti-ionized calcium binding adaptor molecule-1 (Iba1, 1:500 dilution; Wako, Richmond, VA), anti CD-163 (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and anti-CD169 (1:500 dilution; AbD Serotec, Raleigh, NC).

    Mutagenesis:

    Article Title: Reporter-Based Isolation of Developmental Myogenic Progenitors
    Article Snippet: WT and mutant mice expressing a reporter in myogenic progenitors (Table ). .. 1x phosphate buffer saline (PBS) pH 7.4 (ThermoFisher Scientific 10010031) Ethanol 70% (vol/vol) DMEM (High Glucose) (ThermoFisher Scientific 10938025) Penicillin/streptomycin mixtures (P/S) (100X, Omega Scientific PS-20) HEPES 1M (ThermoFisher Scientific 15630080) L-Glutamine 200 mM (ThermoFisher Scientific 25030081) 1x Ca2+ -free Hanks' balanced salt solution (HBSS) (ThermoFisher Scientific 14170070) Bovine serum albumin (BSA) (Sigma A1933) Glucose (Sigma G7021) Magnesium sulfate (MgSO4 ) (Sigma M2643) Sodium hydroxide (NaOH) 1N (Sigma S2770) EDTA disodium salt (Sigma E6635) Ammonium chloride (NH4 Cl) (Sigma A9434) Potassium bicarbonate (KHCO3) (Sigma 60339) EDTA tetrasodium salt hydrate (Sigma E5391) Fetal bovine serum (FBS) (ThermoFisher Scientific 10270106) Collagenase Type V (Sigma C9263) Dispase (ThermoFisher Scientific 17105041) DNAse I (Worthington ) Parafilm Horse serum (ThermoFisher Scientific 16050122) Calf skin collagen (Sigma C8919) ECM gel from Engelbreth-Holm-Swarm murine sarcoma (Sigma E1270) Ultrapure distilled water (ThermoFisher Scientific 10977023) 16% Formaldehyde (w/v), Methanol-free (ThermoFisher Scientific 28906) CAUTION Hazardous in case of eye contact and on ingestion Anti-Pax7 (Developmental Studies Hybridoma Bank AB_528428) Anti-MyoD (Dako M3512) Anti-Myogenin (BD Pharmingen 556358) Anti-Desmin (Sigma SAB5600054) DAPI (diamidino-2-phenylindole) (Sigma 32670) Ice Liquid nitrogen.

    Isolation:

    Article Title: Inactivation of Retinoblastoma Protein (Rb1) in the Oocyte: Evidence That Dysregulated Follicle Growth Drives Ovarian Teratoma Formation in Mice
    Article Snippet: Paragraph title: Isolation and culture of oocytes ... When no evidence of parthenogenesis was observed, we conducted a second series of experiments replicating a previously described culture protocol [ ] in which GV oocytes were collected and cultured in Minimal Essential Medium (MEM) with Earle’s balanced salt solution (Sigma, MO, USA) supplemented with essential amino acids (Sigma, MO, USA), Penicillin and Streptomycin (Gibco, Life Technologies, CA, USA), 0.01 mM Ethylenediaminetetraacetic acid tetrasodium salt hydrate (Tetrasodium EDTA, Sigma, USA), 0.23 mM pyruvic acid (Sigma, USA) and 3% bovine serum albumin (BSA, Sigma, USA).

    Labeling:

    Article Title: Development of a Novel Screening Strategy Designed to Discover a New Class of HIV Drugs
    Article Snippet: FlAsH tag (Lumino Green in-Cell Labeling Kit, #12589-057) was purchased from Invitrogen (Grand Island, NY). .. Dithiothreitol (DTT) (43815), EDTA (E5391), HEPES (H3375), and HIV Protease Substrate 1 (H6660) were obtained from Sigma-Aldrich (St. Louis, MO).

    Purification:

    Article Title: Development of a Novel Screening Strategy Designed to Discover a New Class of HIV Drugs
    Article Snippet: The MA/CAΔ protein substrate and its DNA construct have been described., Purified HIV-1 protease (PR) was purified from Escherichia coli inclusion bodies as described previously. .. Dithiothreitol (DTT) (43815), EDTA (E5391), HEPES (H3375), and HIV Protease Substrate 1 (H6660) were obtained from Sigma-Aldrich (St. Louis, MO).

    Construct:

    Article Title: Development of a Novel Screening Strategy Designed to Discover a New Class of HIV Drugs
    Article Snippet: The MA/CAΔ protein substrate and its DNA construct have been described., Purified HIV-1 protease (PR) was purified from Escherichia coli inclusion bodies as described previously. .. Dithiothreitol (DTT) (43815), EDTA (E5391), HEPES (H3375), and HIV Protease Substrate 1 (H6660) were obtained from Sigma-Aldrich (St. Louis, MO).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Development of a Novel Screening Strategy Designed to Discover a New Class of HIV Drugs
    Article Snippet: Dithiothreitol (DTT) (43815), EDTA (E5391), HEPES (H3375), and HIV Protease Substrate 1 (H6660) were obtained from Sigma-Aldrich (St. Louis, MO). .. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Criterion TGX Stain-Free gel (5678124) and SDS-PAGE loading buffer were obtained from Bio-Rad (Hercules, CA).

    Mouse Assay:

    Article Title: Reporter-Based Isolation of Developmental Myogenic Progenitors
    Article Snippet: We have used WT CD1 or C57BL/6 mice from the Jackson Laboratory or Charles River. .. 1x phosphate buffer saline (PBS) pH 7.4 (ThermoFisher Scientific 10010031) Ethanol 70% (vol/vol) DMEM (High Glucose) (ThermoFisher Scientific 10938025) Penicillin/streptomycin mixtures (P/S) (100X, Omega Scientific PS-20) HEPES 1M (ThermoFisher Scientific 15630080) L-Glutamine 200 mM (ThermoFisher Scientific 25030081) 1x Ca2+ -free Hanks' balanced salt solution (HBSS) (ThermoFisher Scientific 14170070) Bovine serum albumin (BSA) (Sigma A1933) Glucose (Sigma G7021) Magnesium sulfate (MgSO4 ) (Sigma M2643) Sodium hydroxide (NaOH) 1N (Sigma S2770) EDTA disodium salt (Sigma E6635) Ammonium chloride (NH4 Cl) (Sigma A9434) Potassium bicarbonate (KHCO3) (Sigma 60339) EDTA tetrasodium salt hydrate (Sigma E5391) Fetal bovine serum (FBS) (ThermoFisher Scientific 10270106) Collagenase Type V (Sigma C9263) Dispase (ThermoFisher Scientific 17105041) DNAse I (Worthington ) Parafilm Horse serum (ThermoFisher Scientific 16050122) Calf skin collagen (Sigma C8919) ECM gel from Engelbreth-Holm-Swarm murine sarcoma (Sigma E1270) Ultrapure distilled water (ThermoFisher Scientific 10977023) 16% Formaldehyde (w/v), Methanol-free (ThermoFisher Scientific 28906) CAUTION Hazardous in case of eye contact and on ingestion Anti-Pax7 (Developmental Studies Hybridoma Bank AB_528428) Anti-MyoD (Dako M3512) Anti-Myogenin (BD Pharmingen 556358) Anti-Desmin (Sigma SAB5600054) DAPI (diamidino-2-phenylindole) (Sigma 32670) Ice Liquid nitrogen.

    Article Title: Comparative Anatomy of the Mammalian Corneal Subbasal Nerve Plexus
    Article Snippet: .. IHC Staining To enhance tissue permeability and optimize IHC staining, corneas from all animals except mice were first permeabilized in 0.01% hyaluronidase (type IV-S, catalogue #H4272, Sigma-Aldrich, Corp., St. Louis, MO, USA) and 0.1% ethylenediaminetetraacetic acid (EDTA) (catalogue #E5391, Sigma-Aldrich, Corp.) in 0.1 M PBS, pH 5.3. .. Rat and guinea pig corneas were incubated in the permeability reagent overnight at RT, whereas corneas from all larger mammals were incubated for 24 to 48 hours at 37°C.

    SDS Page:

    Article Title: Development of a Novel Screening Strategy Designed to Discover a New Class of HIV Drugs
    Article Snippet: Dithiothreitol (DTT) (43815), EDTA (E5391), HEPES (H3375), and HIV Protease Substrate 1 (H6660) were obtained from Sigma-Aldrich (St. Louis, MO). .. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Criterion TGX Stain-Free gel (5678124) and SDS-PAGE loading buffer were obtained from Bio-Rad (Hercules, CA).

    Affinity Chromatography:

    Article Title: LARGE SCALE PURIFICATION OF BUTYRYLCHOLINESTERASE FROM HUMAN PLASMA SUITABLE FOR INJECTION INTO MONKEYS; A POTENTIAL NEW THERAPEUTIC FOR PROTECTION AGAINST COCAINE AND NERVE AGENT TOXICITY
    Article Snippet: The 100x buffer was made in a glass bottle and contained 152 g EDTA tetrasodium salt (Aldrich E2,629-0, 98% pure) plus 456 ml glacial acetic acid, and 18 g sodium hydroxide in a total volume of 4 L. After 1:100 dilution this buffer had a conductivity of 0.36 mS and a pH of 4.0. .. 50X buffer for affinity chromatography .

    Sample Prep:

    Article Title: A Quadrupole Dalton-based multi-attribute method for product characterization, process development, and quality control of therapeutic proteins
    Article Snippet: Paragraph title: Sample preparation for QDa-based MAM ... The alkylated and denatured antibodies were then diluted using 100 mM sodium phosphate pH 7.0 and 0.1 mM ethylenediaminetetraacetic acid (EDTA, E5391, Sigma) to reduce guanidine concentration to 1.7 M. Lysyl endopeptidase (Lys-C, 125–05061, Wako) was added at 1:5 protease: protein ratio and incubated at 37°C for 4 hours.

    Activation Assay:

    Article Title: Inactivation of Retinoblastoma Protein (Rb1) in the Oocyte: Evidence That Dysregulated Follicle Growth Drives Ovarian Teratoma Formation in Mice
    Article Snippet: The culture condition supported oocyte maturation and parthenogenetic activation because more than 50% of control oocytes treated with 7% ethanol for 5 min developed to 2-cell embryos. .. When no evidence of parthenogenesis was observed, we conducted a second series of experiments replicating a previously described culture protocol [ ] in which GV oocytes were collected and cultured in Minimal Essential Medium (MEM) with Earle’s balanced salt solution (Sigma, MO, USA) supplemented with essential amino acids (Sigma, MO, USA), Penicillin and Streptomycin (Gibco, Life Technologies, CA, USA), 0.01 mM Ethylenediaminetetraacetic acid tetrasodium salt hydrate (Tetrasodium EDTA, Sigma, USA), 0.23 mM pyruvic acid (Sigma, USA) and 3% bovine serum albumin (BSA, Sigma, USA).

    Staining:

    Article Title: The frequencies of micronuclei, nucleoplasmic bridges and nuclear buds as biomarkers of genomic instability in patients with urothelial cell carcinoma
    Article Snippet: Next, the heads of the brushes were individually placed into separate 30 ml yellow-top containers, each of which contained a mixture of the following components: buccal cell buffer (0.01 M Tris-HCl; Sigma T-3253), 0.1 M EDTA tetrasodium salt (Sigma E5391), and 0.02 sodium chloride (Sigma S5886) at pH 7.0. .. Slides were then air dried for 10 min prior to staining with Giemsa (5%).

    Article Title: Development of a Novel Screening Strategy Designed to Discover a New Class of HIV Drugs
    Article Snippet: Dithiothreitol (DTT) (43815), EDTA (E5391), HEPES (H3375), and HIV Protease Substrate 1 (H6660) were obtained from Sigma-Aldrich (St. Louis, MO). .. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Criterion TGX Stain-Free gel (5678124) and SDS-PAGE loading buffer were obtained from Bio-Rad (Hercules, CA).

    Article Title: Comparative Anatomy of the Mammalian Corneal Subbasal Nerve Plexus
    Article Snippet: .. IHC Staining To enhance tissue permeability and optimize IHC staining, corneas from all animals except mice were first permeabilized in 0.01% hyaluronidase (type IV-S, catalogue #H4272, Sigma-Aldrich, Corp., St. Louis, MO, USA) and 0.1% ethylenediaminetetraacetic acid (EDTA) (catalogue #E5391, Sigma-Aldrich, Corp.) in 0.1 M PBS, pH 5.3. .. Rat and guinea pig corneas were incubated in the permeability reagent overnight at RT, whereas corneas from all larger mammals were incubated for 24 to 48 hours at 37°C.

    Permeability:

    Article Title: Comparative Anatomy of the Mammalian Corneal Subbasal Nerve Plexus
    Article Snippet: .. IHC Staining To enhance tissue permeability and optimize IHC staining, corneas from all animals except mice were first permeabilized in 0.01% hyaluronidase (type IV-S, catalogue #H4272, Sigma-Aldrich, Corp., St. Louis, MO, USA) and 0.1% ethylenediaminetetraacetic acid (EDTA) (catalogue #E5391, Sigma-Aldrich, Corp.) in 0.1 M PBS, pH 5.3. .. Rat and guinea pig corneas were incubated in the permeability reagent overnight at RT, whereas corneas from all larger mammals were incubated for 24 to 48 hours at 37°C.

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    Millipore triton x 100 buffer
    Pretreating hippocampal slices with Trolox rescues Aβ 1–42 -induced GLT-1 Triton X-100 detergent-insolubility and ubiquitination.  A , Representative Western blots show GLT-1 levels in protein lysates first subjected to three serial 1% Triton X-100 extractions to remove Triton X-100-soluble proteins. Aβ 1–42  increased detergent-insoluble GLT-1 levels compared with control slices. Trolox reduced GLT-1 detergent-insolubility to control levels. Prestaining with Sypro confirmed equivalent protein loading among lanes.  B , Aβ 1–42  increased Triton X-100-insoluble GLT-1 (left) and was rescued by Trolox pretreatment (Ctrl, Aβ 1–42 , and Aβ 1–42  pretreated 1 h before and during 30 min Aβ 1–42  treatment;  n  = 9, 50, and 18, respectively;  F (2,74)  = 13.605,  p
    Triton X 100 Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore jak2 ip buffer
    Erythropoietin Receptor-Mediated Activation of the <t>JAK2/STAT5,</t> RAS/MAPK, and PI3K/AKT Signaling Pathways is Attenuated by BCR-ABL Kinase Activity in K562 Cells A. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells treated for 1hr with either 100nM dasatinib or 1μM imatinib. Data represents the average ± SD (n=3). JAK2 activation was monitored by immunoprecipitation and activation loop (Y1007) phosphorylation. JAK2 activation was normalized to the level of phospho-Y1007 observed in the “DMSO” condition for each experimental replicate. B. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates from K562 cells after short-term and prolonged BCR-ABL inhibition (100nM dasatinib: 2hrs, 4hrs, 8hrs, 24hrs; 0.2% DMSO, 24hrs) followed by hEPO stimulation (10′). C. Normalized hEPO-mediated JAK2 activation in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed hEPO stimulation (10′). Data is representative of triplicate experimental analysis. JAK2 activation and normalization was performed as in (A). D. Normalized RAS-GTP loading in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed by hEPO stimulation (10′). Data is representative of triplicate experimental analysis. RAS activation was monitored using a RAS-GTP pulldown assay and RAS-GTP levels were normalized to the “DMSO” condition. E. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348. JAK2 activation was monitored and normalized as in (A). F. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348 followed by hEPO stimulation (10′).
    Jak2 Ip Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tris buffer
    Adenovirus-transduced artificial TDP-43 cytoplasmic aggregates have insoluble properties against various detergents. ( a, b ) <t>Sarkosyl-fractionated</t> cell lysates with <t>Tris</t> (T), 1% TritonX-100 (X), 1% sarkosyl (S) and 1 × SDS-sample (P) buffers were examined using phospho (p)-TDP-43 (pSer409/Ser410) ( a ) or C-terminal TDP-43 (405–414) antibody ( b ). ( c, d ) RIPA/urea-fractionated cell lysates with RIPA (R) and urea (U) buffers were examined using pTDP-43 ( c ) or C-terminal TDP-43 (405–414) antibody ( d ). The 74 and 52 kDa bands correspond to phosphorylated DsRed-tagged WT (←pWT) and CTF (←pCTF) TDP-43, respectively ( a, c ). The 72 and 50 kDa bands correspond to non-phosphorylated DsRed-tagged WT (←WT) and CTF (←CTF) TDP-43, respectively. Endogenous rat TDP-43 band at 43 kDa was indicated as arrowhead ( b, d ). ( a-d ) Full length gels and blots are shown.
    Tris Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pretreating hippocampal slices with Trolox rescues Aβ 1–42 -induced GLT-1 Triton X-100 detergent-insolubility and ubiquitination.  A , Representative Western blots show GLT-1 levels in protein lysates first subjected to three serial 1% Triton X-100 extractions to remove Triton X-100-soluble proteins. Aβ 1–42  increased detergent-insoluble GLT-1 levels compared with control slices. Trolox reduced GLT-1 detergent-insolubility to control levels. Prestaining with Sypro confirmed equivalent protein loading among lanes.  B , Aβ 1–42  increased Triton X-100-insoluble GLT-1 (left) and was rescued by Trolox pretreatment (Ctrl, Aβ 1–42 , and Aβ 1–42  pretreated 1 h before and during 30 min Aβ 1–42  treatment;  n  = 9, 50, and 18, respectively;  F (2,74)  = 13.605,  p

    Journal: The Journal of Neuroscience

    Article Title: Amyloid-?1–42 Slows Clearance of Synaptically Released Glutamate by Mislocalizing Astrocytic GLT-1

    doi: 10.1523/JNEUROSCI.5274-12.2013

    Figure Lengend Snippet: Pretreating hippocampal slices with Trolox rescues Aβ 1–42 -induced GLT-1 Triton X-100 detergent-insolubility and ubiquitination. A , Representative Western blots show GLT-1 levels in protein lysates first subjected to three serial 1% Triton X-100 extractions to remove Triton X-100-soluble proteins. Aβ 1–42 increased detergent-insoluble GLT-1 levels compared with control slices. Trolox reduced GLT-1 detergent-insolubility to control levels. Prestaining with Sypro confirmed equivalent protein loading among lanes. B , Aβ 1–42 increased Triton X-100-insoluble GLT-1 (left) and was rescued by Trolox pretreatment (Ctrl, Aβ 1–42 , and Aβ 1–42 pretreated 1 h before and during 30 min Aβ 1–42 treatment; n = 9, 50, and 18, respectively; F (2,74) = 13.605, p

    Article Snippet: Protein lysates from slices and cultured astrocytes were prepared using 1% Triton X-100 buffer [in m m : 20 Tris, 1 EDTA, 0.5 EGTA, 250 sucrose, Protease Inhibitor Cocktail (EMD Millipore)].

    Techniques: Western Blot

    Erythropoietin Receptor-Mediated Activation of the JAK2/STAT5, RAS/MAPK, and PI3K/AKT Signaling Pathways is Attenuated by BCR-ABL Kinase Activity in K562 Cells A. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells treated for 1hr with either 100nM dasatinib or 1μM imatinib. Data represents the average ± SD (n=3). JAK2 activation was monitored by immunoprecipitation and activation loop (Y1007) phosphorylation. JAK2 activation was normalized to the level of phospho-Y1007 observed in the “DMSO” condition for each experimental replicate. B. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates from K562 cells after short-term and prolonged BCR-ABL inhibition (100nM dasatinib: 2hrs, 4hrs, 8hrs, 24hrs; 0.2% DMSO, 24hrs) followed by hEPO stimulation (10′). C. Normalized hEPO-mediated JAK2 activation in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed hEPO stimulation (10′). Data is representative of triplicate experimental analysis. JAK2 activation and normalization was performed as in (A). D. Normalized RAS-GTP loading in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed by hEPO stimulation (10′). Data is representative of triplicate experimental analysis. RAS activation was monitored using a RAS-GTP pulldown assay and RAS-GTP levels were normalized to the “DMSO” condition. E. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348. JAK2 activation was monitored and normalized as in (A). F. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348 followed by hEPO stimulation (10′).

    Journal: Cancer discovery

    Article Title: MEK-Dependent Negative Feedback Underlies BCR-ABL-Mediated Oncogene Addiction

    doi: 10.1158/2159-8290.CD-13-0235

    Figure Lengend Snippet: Erythropoietin Receptor-Mediated Activation of the JAK2/STAT5, RAS/MAPK, and PI3K/AKT Signaling Pathways is Attenuated by BCR-ABL Kinase Activity in K562 Cells A. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells treated for 1hr with either 100nM dasatinib or 1μM imatinib. Data represents the average ± SD (n=3). JAK2 activation was monitored by immunoprecipitation and activation loop (Y1007) phosphorylation. JAK2 activation was normalized to the level of phospho-Y1007 observed in the “DMSO” condition for each experimental replicate. B. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates from K562 cells after short-term and prolonged BCR-ABL inhibition (100nM dasatinib: 2hrs, 4hrs, 8hrs, 24hrs; 0.2% DMSO, 24hrs) followed by hEPO stimulation (10′). C. Normalized hEPO-mediated JAK2 activation in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed hEPO stimulation (10′). Data is representative of triplicate experimental analysis. JAK2 activation and normalization was performed as in (A). D. Normalized RAS-GTP loading in K562 cells after short-term (1hr) and prolonged (24hrs) 100nM dasatinib treatment followed by hEPO stimulation (10′). Data is representative of triplicate experimental analysis. RAS activation was monitored using a RAS-GTP pulldown assay and RAS-GTP levels were normalized to the “DMSO” condition. E. Normalized hEPO-mediated (10′ stimulation) JAK2 activation in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348. JAK2 activation was monitored and normalized as in (A). F. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of whole cell lysates in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 1μM imatinib, 500nM TG101348, dasatinib/TG101348, or imatinib/TG101348 followed by hEPO stimulation (10′).

    Article Snippet: Cells were lysed in JAK2 IP buffer (50mM Tris pH 7.6, 100mM NaCl, 1mM EDTA, 1mM EGTA, 0.5% NP40, 0.1% Triton) or RAS IP buffer (50mM Tris pH 7.5, 125mM NaCl, 6.5mM MgCl2 , 5% glycerol, 0.2% NP40) supplemented with 1% protease and 1% phosphatase inhibitors (Calbiochem).

    Techniques: Activation Assay, Activity Assay, Immunoprecipitation, Western Blot, Inhibition

    Global Gene Expression Analysis of Dasatinib Treated K562 Cells Identifies Candidate Mediators Responsible for the Attenuation of Myeloid GF-R Signaling in CML Cells A. ). The heat map at the bottom highlights a few of the genes with increased expression following dasatinib treatment in K562 cells. B. Quantitative PCR (qPCR) analysis of potential negative feedback genes in K562 cells treated with dasatinib (100nM), imatinib (1μM), or PD0325901 (500nM). The average fold expression change (2 (−ΔΔCt) ) and standard deviation (SD fold-change ) for each gene is represented (n=3). C. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of RAS and ERK activity before and after hEPO-stimulation (10′) in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 500nM PD0325901, or dasatinib/PD0325901. RAS activity was monitored using a RAS-GTP pulldown assay. D. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of JAK2 and STAT5 activity before and after hEPO-stimulation in K562 cells treated under the same experimental conditions as (A). JAK2 activation was determined by immunoprecipitation and activation loop (Y1007) phosphorylation.

    Journal: Cancer discovery

    Article Title: MEK-Dependent Negative Feedback Underlies BCR-ABL-Mediated Oncogene Addiction

    doi: 10.1158/2159-8290.CD-13-0235

    Figure Lengend Snippet: Global Gene Expression Analysis of Dasatinib Treated K562 Cells Identifies Candidate Mediators Responsible for the Attenuation of Myeloid GF-R Signaling in CML Cells A. ). The heat map at the bottom highlights a few of the genes with increased expression following dasatinib treatment in K562 cells. B. Quantitative PCR (qPCR) analysis of potential negative feedback genes in K562 cells treated with dasatinib (100nM), imatinib (1μM), or PD0325901 (500nM). The average fold expression change (2 (−ΔΔCt) ) and standard deviation (SD fold-change ) for each gene is represented (n=3). C. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of RAS and ERK activity before and after hEPO-stimulation (10′) in K562 cells pretreated for 24hrs with 0.2% DMSO, 100nM dasatinib, 500nM PD0325901, or dasatinib/PD0325901. RAS activity was monitored using a RAS-GTP pulldown assay. D. Upper: Line diagram representation of duration of serum starve, kinase inhibitor treatment, and growth-factor stimulation. Lower: Western immunoblot analysis of JAK2 and STAT5 activity before and after hEPO-stimulation in K562 cells treated under the same experimental conditions as (A). JAK2 activation was determined by immunoprecipitation and activation loop (Y1007) phosphorylation.

    Article Snippet: Cells were lysed in JAK2 IP buffer (50mM Tris pH 7.6, 100mM NaCl, 1mM EDTA, 1mM EGTA, 0.5% NP40, 0.1% Triton) or RAS IP buffer (50mM Tris pH 7.5, 125mM NaCl, 6.5mM MgCl2 , 5% glycerol, 0.2% NP40) supplemented with 1% protease and 1% phosphatase inhibitors (Calbiochem).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Western Blot, Activity Assay, Activation Assay, Immunoprecipitation

    Adenovirus-transduced artificial TDP-43 cytoplasmic aggregates have insoluble properties against various detergents. ( a, b ) Sarkosyl-fractionated cell lysates with Tris (T), 1% TritonX-100 (X), 1% sarkosyl (S) and 1 × SDS-sample (P) buffers were examined using phospho (p)-TDP-43 (pSer409/Ser410) ( a ) or C-terminal TDP-43 (405–414) antibody ( b ). ( c, d ) RIPA/urea-fractionated cell lysates with RIPA (R) and urea (U) buffers were examined using pTDP-43 ( c ) or C-terminal TDP-43 (405–414) antibody ( d ). The 74 and 52 kDa bands correspond to phosphorylated DsRed-tagged WT (←pWT) and CTF (←pCTF) TDP-43, respectively ( a, c ). The 72 and 50 kDa bands correspond to non-phosphorylated DsRed-tagged WT (←WT) and CTF (←CTF) TDP-43, respectively. Endogenous rat TDP-43 band at 43 kDa was indicated as arrowhead ( b, d ). ( a-d ) Full length gels and blots are shown.

    Journal: PLoS ONE

    Article Title: Formation and spreading of TDP-43 aggregates in cultured neuronal and glial cells demonstrated by time-lapse imaging

    doi: 10.1371/journal.pone.0179375

    Figure Lengend Snippet: Adenovirus-transduced artificial TDP-43 cytoplasmic aggregates have insoluble properties against various detergents. ( a, b ) Sarkosyl-fractionated cell lysates with Tris (T), 1% TritonX-100 (X), 1% sarkosyl (S) and 1 × SDS-sample (P) buffers were examined using phospho (p)-TDP-43 (pSer409/Ser410) ( a ) or C-terminal TDP-43 (405–414) antibody ( b ). ( c, d ) RIPA/urea-fractionated cell lysates with RIPA (R) and urea (U) buffers were examined using pTDP-43 ( c ) or C-terminal TDP-43 (405–414) antibody ( d ). The 74 and 52 kDa bands correspond to phosphorylated DsRed-tagged WT (←pWT) and CTF (←pCTF) TDP-43, respectively ( a, c ). The 72 and 50 kDa bands correspond to non-phosphorylated DsRed-tagged WT (←WT) and CTF (←CTF) TDP-43, respectively. Endogenous rat TDP-43 band at 43 kDa was indicated as arrowhead ( b, d ). ( a-d ) Full length gels and blots are shown.

    Article Snippet: Cell fractionation For sarkosyl fractionation, cells were lysed and sonicated in Tris buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA and 5 mM EGTA) containing phosphatase inhibitors (5 mM NaF and 2 mM Na3 VO4 ) and 1× protease inhibitor cocktail (Millipore, Billerica, MA, USA), followed by centrifugation at 160,000 × g for 20 min at 4°C.

    Techniques: