mgcl ²  (Thermo Fisher)


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  • 99
    Name:
    MgCl2 1 M
    Description:
    Molecular biology grade Ambion 1 M MgCl2 solution is supplied in one bottle containing 100 mL The solution is certified RNase free economical and ready to use Due to the ubiquitous presence of RNases manufacturing products for use with RNA is especially challenging Ambion s nuclease free reagents and buffers are manufactured in facilities specifically designed to prevent the introduction of nucleases Highly sensitive RNase assays are performed at several different stages of the manufacturing process to ensure the highest quality These reagents are rigorously tested for contaminating nonspecific endonuclease exonuclease and RNase activity
    Catalog Number:
    am9530g
    Price:
    None
    Category:
    Lab Reagents and Chemicals
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    Structured Review

    Thermo Fisher mgcl ²
    Molecular biology grade Ambion 1 M MgCl2 solution is supplied in one bottle containing 100 mL The solution is certified RNase free economical and ready to use Due to the ubiquitous presence of RNases manufacturing products for use with RNA is especially challenging Ambion s nuclease free reagents and buffers are manufactured in facilities specifically designed to prevent the introduction of nucleases Highly sensitive RNase assays are performed at several different stages of the manufacturing process to ensure the highest quality These reagents are rigorously tested for contaminating nonspecific endonuclease exonuclease and RNase activity
    https://www.bioz.com/result/mgcl ²/product/Thermo Fisher
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    mgcl ² - by Bioz Stars, 2020-07
    99/100 stars

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    Related Articles

    Methylation Sequencing:

    Article Title: Histone-Acetylated Control of Fibroblast Growth Factor Receptor 2 Intron 2 Polymorphisms and Isoform Splicing in Breast Cancer
    Article Snippet: .. Two rounds of PCRs were carried out for bisulfite sequencing, both for 10 min at 95 C followed by 40 cycles of 30 sec at 95 C, 45 sec at Tm and 45 sec at 72 C, followed by a 10-min extension at 72 C in a reaction mixture containing 1.5 m m MgCl2 , 0.2 m m dNTP, 0.2 m m of each primer, and 0.25 U/10 μl of Ampli Taq Gold polymerase (Applied Biosystems). .. Final PCR products were cut from 2.0% agarose gels, extracted, and cloned into the TA cloning system (Invitrogen) for automated sequencing.

    Synthesized:

    Article Title: Adrenomedullin2 (ADM2)/Intermedin (IMD) in Rat Ovary: Changes in Estrous Cycle and Pregnancy and Its Role in Ovulation and Steroidogenesis 1
    Article Snippet: .. Using total RNA, first-strand complementary DNA (cDNA) was synthesized by reverse transcription in a 20 μl reaction volume containing 1× PCR buffer, 2 μg RNA, 5 mM MgCl2 , 1 mM dNTP mixture, and random primers as described by the supplier (Ambion). ..

    Overlay Assay:

    Article Title: Transgenic Overexpression of LARGE Induces ?-Dystroglycan Hyperglycosylation in Skeletal and Cardiac Muscle
    Article Snippet: .. For the laminin overlay assay, nitrocellulose membranes were blocked for 1 hour in laminin binding buffer (LBB: 10 mM triethanolamine, 140 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, pH 7.6) containing 5% non-fat dry milk followed by incubation of mouse Engelbreth-Holm-Swarm laminin (Invitrogen,USA) overnight at 4°C in LBB. .. Membranes were washed and incubated with anti rabbit laminin (Sigma, USA) followed by HRP-anti rabbit IgG (Jackson ImmunoResearch, USA).

    Article Title: The transgenic expression of LARGE exacerbates the muscle phenotype of dystroglycanopathy mice
    Article Snippet: .. For the laminin overlay assay, nitrocellulose membranes were blocked for 1 hour in laminin binding buffer (LBB: 10 mM triethanolamine, 140 mM NaCl, 1 mM MgCl2 , 1 mM CaCl2 , pH 7.6) containing 5% non-fat dry milk followed by incubation of mouse Engelbreth-Holm-Swarm laminin (Invitrogen,USA) overnight at 4°C in LBB. .. Membranes were washed and incubated with anti rabbit laminin (Sigma, USA) followed by HRP-anti rabbit IgG (Jackson ImmunoResearch, USA).

    Article Title: Prenatal muscle development in a mouse model for the secondary dystroglycanopathies
    Article Snippet: .. For the laminin overlay assay, PVDF membranes were blocked for 1 h in laminin-binding buffer (LBB; 10 mM triethanolamine, 140 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, pH 7.6) containing 5 % non-fat dry milk followed by incubation of 1 μg/ml mouse Engelbreth–Holm–Swarm (EHS) laminin protein (Invitrogen) in LBB overnight on a roller at 4 °C. .. The membranes were washed and incubated with rabbit anti-laminin (Sigma) followed by HRP-anti-rabbit IgG (Jackson ImmunoResearch).

    Size-exclusion Chromatography:

    Article Title: Histone-Acetylated Control of Fibroblast Growth Factor Receptor 2 Intron 2 Polymorphisms and Isoform Splicing in Breast Cancer
    Article Snippet: .. Two rounds of PCRs were carried out for bisulfite sequencing, both for 10 min at 95 C followed by 40 cycles of 30 sec at 95 C, 45 sec at Tm and 45 sec at 72 C, followed by a 10-min extension at 72 C in a reaction mixture containing 1.5 m m MgCl2 , 0.2 m m dNTP, 0.2 m m of each primer, and 0.25 U/10 μl of Ampli Taq Gold polymerase (Applied Biosystems). .. Final PCR products were cut from 2.0% agarose gels, extracted, and cloned into the TA cloning system (Invitrogen) for automated sequencing.

    Concentration Assay:

    Article Title: Pumilio Homologue from Saprolegnia parasitica Specifically Expressed in Undifferentiated Spore Cysts
    Article Snippet: .. Each 12-μl PCR mixture contained cDNA corresponding to 25 ng of RNA, 1.5 mM MgCl2 , a 0.2 mM concentration of each deoxynucleoside triphosphate, a 0.4 mM concentration of each primer, and 1 U of Taq polymerase (Gibco) mixed with Platinum Antitaq antibodies (Gibco). ..

    Incubation:

    Article Title: Transgenic Overexpression of LARGE Induces ?-Dystroglycan Hyperglycosylation in Skeletal and Cardiac Muscle
    Article Snippet: .. For the laminin overlay assay, nitrocellulose membranes were blocked for 1 hour in laminin binding buffer (LBB: 10 mM triethanolamine, 140 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, pH 7.6) containing 5% non-fat dry milk followed by incubation of mouse Engelbreth-Holm-Swarm laminin (Invitrogen,USA) overnight at 4°C in LBB. .. Membranes were washed and incubated with anti rabbit laminin (Sigma, USA) followed by HRP-anti rabbit IgG (Jackson ImmunoResearch, USA).

    Article Title: Down-Regulation of Telomerase Activity and Activation of Caspase-3 Are Responsible for Tanshinone I-Induced Apoptosis in Monocyte Leukemia Cells in Vitro
    Article Snippet: .. To generate hTERT cDNA, a 20-μL reaction containing 1μg of total RNA, 25 mmol/L MgCl2 , 10 × PCR buffer, 10 mmol/LDntp, 0.1 MDTT, 1 unit/μL of RNase inhibitor, 200 units of murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, Md., USA), and 2.5 μmol/L of random hexamers was incubated for 10 min at 65 °C. ..

    Article Title: The transgenic expression of LARGE exacerbates the muscle phenotype of dystroglycanopathy mice
    Article Snippet: .. For the laminin overlay assay, nitrocellulose membranes were blocked for 1 hour in laminin binding buffer (LBB: 10 mM triethanolamine, 140 mM NaCl, 1 mM MgCl2 , 1 mM CaCl2 , pH 7.6) containing 5% non-fat dry milk followed by incubation of mouse Engelbreth-Holm-Swarm laminin (Invitrogen,USA) overnight at 4°C in LBB. .. Membranes were washed and incubated with anti rabbit laminin (Sigma, USA) followed by HRP-anti rabbit IgG (Jackson ImmunoResearch, USA).

    Article Title: Prenatal muscle development in a mouse model for the secondary dystroglycanopathies
    Article Snippet: .. For the laminin overlay assay, PVDF membranes were blocked for 1 h in laminin-binding buffer (LBB; 10 mM triethanolamine, 140 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, pH 7.6) containing 5 % non-fat dry milk followed by incubation of 1 μg/ml mouse Engelbreth–Holm–Swarm (EHS) laminin protein (Invitrogen) in LBB overnight on a roller at 4 °C. .. The membranes were washed and incubated with rabbit anti-laminin (Sigma) followed by HRP-anti-rabbit IgG (Jackson ImmunoResearch).

    Polymerase Chain Reaction:

    Article Title: Adrenomedullin2 (ADM2)/Intermedin (IMD) in Rat Ovary: Changes in Estrous Cycle and Pregnancy and Its Role in Ovulation and Steroidogenesis 1
    Article Snippet: .. Using total RNA, first-strand complementary DNA (cDNA) was synthesized by reverse transcription in a 20 μl reaction volume containing 1× PCR buffer, 2 μg RNA, 5 mM MgCl2 , 1 mM dNTP mixture, and random primers as described by the supplier (Ambion). ..

    Article Title: Down-Regulation of Telomerase Activity and Activation of Caspase-3 Are Responsible for Tanshinone I-Induced Apoptosis in Monocyte Leukemia Cells in Vitro
    Article Snippet: .. To generate hTERT cDNA, a 20-μL reaction containing 1μg of total RNA, 25 mmol/L MgCl2 , 10 × PCR buffer, 10 mmol/LDntp, 0.1 MDTT, 1 unit/μL of RNase inhibitor, 200 units of murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, Md., USA), and 2.5 μmol/L of random hexamers was incubated for 10 min at 65 °C. ..

    Article Title: Pumilio Homologue from Saprolegnia parasitica Specifically Expressed in Undifferentiated Spore Cysts
    Article Snippet: .. Each 12-μl PCR mixture contained cDNA corresponding to 25 ng of RNA, 1.5 mM MgCl2 , a 0.2 mM concentration of each deoxynucleoside triphosphate, a 0.4 mM concentration of each primer, and 1 U of Taq polymerase (Gibco) mixed with Platinum Antitaq antibodies (Gibco). ..

    Binding Assay:

    Article Title: Transgenic Overexpression of LARGE Induces ?-Dystroglycan Hyperglycosylation in Skeletal and Cardiac Muscle
    Article Snippet: .. For the laminin overlay assay, nitrocellulose membranes were blocked for 1 hour in laminin binding buffer (LBB: 10 mM triethanolamine, 140 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, pH 7.6) containing 5% non-fat dry milk followed by incubation of mouse Engelbreth-Holm-Swarm laminin (Invitrogen,USA) overnight at 4°C in LBB. .. Membranes were washed and incubated with anti rabbit laminin (Sigma, USA) followed by HRP-anti rabbit IgG (Jackson ImmunoResearch, USA).

    Article Title: The transgenic expression of LARGE exacerbates the muscle phenotype of dystroglycanopathy mice
    Article Snippet: .. For the laminin overlay assay, nitrocellulose membranes were blocked for 1 hour in laminin binding buffer (LBB: 10 mM triethanolamine, 140 mM NaCl, 1 mM MgCl2 , 1 mM CaCl2 , pH 7.6) containing 5% non-fat dry milk followed by incubation of mouse Engelbreth-Holm-Swarm laminin (Invitrogen,USA) overnight at 4°C in LBB. .. Membranes were washed and incubated with anti rabbit laminin (Sigma, USA) followed by HRP-anti rabbit IgG (Jackson ImmunoResearch, USA).

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  • 85
    Thermo Fisher hsp90 binding buffer
    Schematic representation of the <t>Hsp90</t> occupancy assay. A drug-treated cancer cell lysate (sample) was passed over a gel filtration spin column at 4 °C, and the sample was split into two aliquots. In one sample, total Hsp90 was determined by quantitative immunoblotting using separate antibodies to detect both Hsp90α and Hsp90β isoforms. In the second sample, open Hsp90 binding sites were titrated with [ 3 H]17-AAG at 4 °C. Percent of Hsp90 occupancy was calculated from a ratio of Hsp90 open binding sites to total Hsp90.
    Hsp90 Binding Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hsp90 binding buffer/product/Thermo Fisher
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hsp90 binding buffer - by Bioz Stars, 2020-07
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    92
    Thermo Fisher mgcl
    Schematic representation of the <t>Hsp90</t> occupancy assay. A drug-treated cancer cell lysate (sample) was passed over a gel filtration spin column at 4 °C, and the sample was split into two aliquots. In one sample, total Hsp90 was determined by quantitative immunoblotting using separate antibodies to detect both Hsp90α and Hsp90β isoforms. In the second sample, open Hsp90 binding sites were titrated with [ 3 H]17-AAG at 4 °C. Percent of Hsp90 occupancy was calculated from a ratio of Hsp90 open binding sites to total Hsp90.
    Mgcl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mgcl/product/Thermo Fisher
    Average 92 stars, based on 116 article reviews
    Price from $9.99 to $1999.99
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    86
    Thermo Fisher 8 mm mgcl
    Schematic representation of the <t>Hsp90</t> occupancy assay. A drug-treated cancer cell lysate (sample) was passed over a gel filtration spin column at 4 °C, and the sample was split into two aliquots. In one sample, total Hsp90 was determined by quantitative immunoblotting using separate antibodies to detect both Hsp90α and Hsp90β isoforms. In the second sample, open Hsp90 binding sites were titrated with [ 3 H]17-AAG at 4 °C. Percent of Hsp90 occupancy was calculated from a ratio of Hsp90 open binding sites to total Hsp90.
    8 Mm Mgcl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/8 mm mgcl/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
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    8 mm mgcl - by Bioz Stars, 2020-07
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    93
    Thermo Fisher buffer
    Schematic representation of the <t>Hsp90</t> occupancy assay. A drug-treated cancer cell lysate (sample) was passed over a gel filtration spin column at 4 °C, and the sample was split into two aliquots. In one sample, total Hsp90 was determined by quantitative immunoblotting using separate antibodies to detect both Hsp90α and Hsp90β isoforms. In the second sample, open Hsp90 binding sites were titrated with [ 3 H]17-AAG at 4 °C. Percent of Hsp90 occupancy was calculated from a ratio of Hsp90 open binding sites to total Hsp90.
    Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/buffer/product/Thermo Fisher
    Average 93 stars, based on 86 article reviews
    Price from $9.99 to $1999.99
    buffer - by Bioz Stars, 2020-07
    93/100 stars
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    Schematic representation of the Hsp90 occupancy assay. A drug-treated cancer cell lysate (sample) was passed over a gel filtration spin column at 4 °C, and the sample was split into two aliquots. In one sample, total Hsp90 was determined by quantitative immunoblotting using separate antibodies to detect both Hsp90α and Hsp90β isoforms. In the second sample, open Hsp90 binding sites were titrated with [ 3 H]17-AAG at 4 °C. Percent of Hsp90 occupancy was calculated from a ratio of Hsp90 open binding sites to total Hsp90.

    Journal: The Journal of Biological Chemistry

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    doi: 10.1074/jbc.M110.141580

    Figure Lengend Snippet: Schematic representation of the Hsp90 occupancy assay. A drug-treated cancer cell lysate (sample) was passed over a gel filtration spin column at 4 °C, and the sample was split into two aliquots. In one sample, total Hsp90 was determined by quantitative immunoblotting using separate antibodies to detect both Hsp90α and Hsp90β isoforms. In the second sample, open Hsp90 binding sites were titrated with [ 3 H]17-AAG at 4 °C. Percent of Hsp90 occupancy was calculated from a ratio of Hsp90 open binding sites to total Hsp90.

    Article Snippet: Binding Kinetics for Purified Hsp90 and Hsp90 from Cancer Cell Lysates For dissociation off-rate determinations, a [3 H]17-AAG·Hsp90 complex was formed by incubating radiolabeled 17-AAG (200 nm ) with purified Hsp90 (100 nm ) or SK-BR-3 lysates (∼100 nm Hsp90 as determined by quantitative immunoblotting) at 4 °C overnight in Hsp90 binding buffer (20 mm Hepes, pH 7.3, 1 mm EDTA, 100 mm KCl, 5 mm MgCl, 0.01% (v/v) Nonidet P-40, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific), 0.5 mg/ml bovine gamma globulin, and protease inhibitor mixture (Roche Diagnostics GmbH).

    Techniques: Filtration, Binding Assay

    Hsp90 open binding sites can be titrated with [ 3 H]17-AAG. Recombinant human Hsp90β protein (100 n m ) was incubated with increasing concentrations of unlabeled 17-AAG overnight at 4 °C followed by removal of unbound 17-AAG with prechilled size exclusion columns. Free Hsp90 sites were titrated with [ 3 H]17-AAG at 4 °C as described under “Experimental Procedures.” The data from triplicate binding experiments were fit to a four parameter logistic equation.

    Journal: The Journal of Biological Chemistry

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    doi: 10.1074/jbc.M110.141580

    Figure Lengend Snippet: Hsp90 open binding sites can be titrated with [ 3 H]17-AAG. Recombinant human Hsp90β protein (100 n m ) was incubated with increasing concentrations of unlabeled 17-AAG overnight at 4 °C followed by removal of unbound 17-AAG with prechilled size exclusion columns. Free Hsp90 sites were titrated with [ 3 H]17-AAG at 4 °C as described under “Experimental Procedures.” The data from triplicate binding experiments were fit to a four parameter logistic equation.

    Article Snippet: Binding Kinetics for Purified Hsp90 and Hsp90 from Cancer Cell Lysates For dissociation off-rate determinations, a [3 H]17-AAG·Hsp90 complex was formed by incubating radiolabeled 17-AAG (200 nm ) with purified Hsp90 (100 nm ) or SK-BR-3 lysates (∼100 nm Hsp90 as determined by quantitative immunoblotting) at 4 °C overnight in Hsp90 binding buffer (20 mm Hepes, pH 7.3, 1 mm EDTA, 100 mm KCl, 5 mm MgCl, 0.01% (v/v) Nonidet P-40, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific), 0.5 mg/ml bovine gamma globulin, and protease inhibitor mixture (Roche Diagnostics GmbH).

    Techniques: Binding Assay, Recombinant, Incubation

    Determination of Hsp90 occupancy in living cells. H1650 cells were incubated with increasing concentrations of IPI-504 for 6 h at 37 °C. Total Hsp90 protein levels were determined by quantitative immunoblotting using separate anti Hsp90α and Hsp90β antibodies and recombinant proteins as internal standards ( A ). Percent Hsp90 occupancy was determined by titration of open binding sites at 4 °C and total Hsp90 ( B and C ). Data are from a representative experiment with n = 2.

    Journal: The Journal of Biological Chemistry

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    doi: 10.1074/jbc.M110.141580

    Figure Lengend Snippet: Determination of Hsp90 occupancy in living cells. H1650 cells were incubated with increasing concentrations of IPI-504 for 6 h at 37 °C. Total Hsp90 protein levels were determined by quantitative immunoblotting using separate anti Hsp90α and Hsp90β antibodies and recombinant proteins as internal standards ( A ). Percent Hsp90 occupancy was determined by titration of open binding sites at 4 °C and total Hsp90 ( B and C ). Data are from a representative experiment with n = 2.

    Article Snippet: Binding Kinetics for Purified Hsp90 and Hsp90 from Cancer Cell Lysates For dissociation off-rate determinations, a [3 H]17-AAG·Hsp90 complex was formed by incubating radiolabeled 17-AAG (200 nm ) with purified Hsp90 (100 nm ) or SK-BR-3 lysates (∼100 nm Hsp90 as determined by quantitative immunoblotting) at 4 °C overnight in Hsp90 binding buffer (20 mm Hepes, pH 7.3, 1 mm EDTA, 100 mm KCl, 5 mm MgCl, 0.01% (v/v) Nonidet P-40, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific), 0.5 mg/ml bovine gamma globulin, and protease inhibitor mixture (Roche Diagnostics GmbH).

    Techniques: Incubation, Recombinant, Titration, Binding Assay

    Dissociation of [ 3 H]17-AAG from purified Hsp90 and SK-BR-3 lysates is highly temperature-dependent. Purified Hela Hsp90 (100 n m ) or lysate Hsp90·[ 3 H]17-AAG complexes were formed as described under “Experimental Procedures” and passed over size exclusion spin columns. Column eluates were incubated with 10 μ m cold 17-AAG, and samples were removed at different time points. A loss of bound radioactive 17-AAG counts from Hsp90 was measured at both 4 and 37 °C ( A ) and Hsp90 in SK-BR-3 cancer cell lysate at 4 °C ( B ). The data were fit to a monoexponential decay equation.

    Journal: The Journal of Biological Chemistry

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    doi: 10.1074/jbc.M110.141580

    Figure Lengend Snippet: Dissociation of [ 3 H]17-AAG from purified Hsp90 and SK-BR-3 lysates is highly temperature-dependent. Purified Hela Hsp90 (100 n m ) or lysate Hsp90·[ 3 H]17-AAG complexes were formed as described under “Experimental Procedures” and passed over size exclusion spin columns. Column eluates were incubated with 10 μ m cold 17-AAG, and samples were removed at different time points. A loss of bound radioactive 17-AAG counts from Hsp90 was measured at both 4 and 37 °C ( A ) and Hsp90 in SK-BR-3 cancer cell lysate at 4 °C ( B ). The data were fit to a monoexponential decay equation.

    Article Snippet: Binding Kinetics for Purified Hsp90 and Hsp90 from Cancer Cell Lysates For dissociation off-rate determinations, a [3 H]17-AAG·Hsp90 complex was formed by incubating radiolabeled 17-AAG (200 nm ) with purified Hsp90 (100 nm ) or SK-BR-3 lysates (∼100 nm Hsp90 as determined by quantitative immunoblotting) at 4 °C overnight in Hsp90 binding buffer (20 mm Hepes, pH 7.3, 1 mm EDTA, 100 mm KCl, 5 mm MgCl, 0.01% (v/v) Nonidet P-40, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific), 0.5 mg/ml bovine gamma globulin, and protease inhibitor mixture (Roche Diagnostics GmbH).

    Techniques: Purification, Incubation

    Hsp90 occupancy is a better predictor of in vivo pharmacodynamic effects by IPI-504 than tumor or plasma PK. H1650 tumor-bearing mice were treated with a single dose of 100 mg/kg intravenous IPI-504. Tumors and blood plasma were harvested at designated time points post dose. Drug levels of Hsp90 active species (IPI-504, 17-AAG, and 17-AG) were quantified by LC-MS/MS in plasma and in tumor ( A ). EGFR protein levels ( B ) and Hsp90 occupancy ( C ) were measured in tumor tissue. Data are expressed as averages ± S.D. (vehicle ( veh ), n = 2; and 1–48 h, n = 3).

    Journal: The Journal of Biological Chemistry

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    doi: 10.1074/jbc.M110.141580

    Figure Lengend Snippet: Hsp90 occupancy is a better predictor of in vivo pharmacodynamic effects by IPI-504 than tumor or plasma PK. H1650 tumor-bearing mice were treated with a single dose of 100 mg/kg intravenous IPI-504. Tumors and blood plasma were harvested at designated time points post dose. Drug levels of Hsp90 active species (IPI-504, 17-AAG, and 17-AG) were quantified by LC-MS/MS in plasma and in tumor ( A ). EGFR protein levels ( B ) and Hsp90 occupancy ( C ) were measured in tumor tissue. Data are expressed as averages ± S.D. (vehicle ( veh ), n = 2; and 1–48 h, n = 3).

    Article Snippet: Binding Kinetics for Purified Hsp90 and Hsp90 from Cancer Cell Lysates For dissociation off-rate determinations, a [3 H]17-AAG·Hsp90 complex was formed by incubating radiolabeled 17-AAG (200 nm ) with purified Hsp90 (100 nm ) or SK-BR-3 lysates (∼100 nm Hsp90 as determined by quantitative immunoblotting) at 4 °C overnight in Hsp90 binding buffer (20 mm Hepes, pH 7.3, 1 mm EDTA, 100 mm KCl, 5 mm MgCl, 0.01% (v/v) Nonidet P-40, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific), 0.5 mg/ml bovine gamma globulin, and protease inhibitor mixture (Roche Diagnostics GmbH).

    Techniques: In Vivo, Mouse Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Hsp90 occupancy correlates with antitumor activity of IPI-504 in a xenograft model of NSCLC. H1650 tumor bearing mice were treated with vehicle (●), 25 mg/kg (□), 50 mg/kg (▾), or 100 mg/kg (○) of IPI-504 (IP, twice weekly) and tumor growth was assessed by caliper measurement ( A ). Data are expressed as means and standard error ( n = 10 per arm). A separate group of H1650 tumor bearing mice was treated with a single dose of 25, 50, or 100 mg/kg of IPI-504 and sacrificed 2 h post dose. Hsp90 occupancy was determined as described and plotted against tumor size (mm 3 ) measured on day 50 of drug treatment ( B ). Data are expressed as averages with standard deviation ( n = 2 ( x -axis)) and as means with standard error ( n = 10 ( y -axis)). The points were fit to a linear least squares regression equation with a calculated R 2 = 0.98.

    Journal: The Journal of Biological Chemistry

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    doi: 10.1074/jbc.M110.141580

    Figure Lengend Snippet: Hsp90 occupancy correlates with antitumor activity of IPI-504 in a xenograft model of NSCLC. H1650 tumor bearing mice were treated with vehicle (●), 25 mg/kg (□), 50 mg/kg (▾), or 100 mg/kg (○) of IPI-504 (IP, twice weekly) and tumor growth was assessed by caliper measurement ( A ). Data are expressed as means and standard error ( n = 10 per arm). A separate group of H1650 tumor bearing mice was treated with a single dose of 25, 50, or 100 mg/kg of IPI-504 and sacrificed 2 h post dose. Hsp90 occupancy was determined as described and plotted against tumor size (mm 3 ) measured on day 50 of drug treatment ( B ). Data are expressed as averages with standard deviation ( n = 2 ( x -axis)) and as means with standard error ( n = 10 ( y -axis)). The points were fit to a linear least squares regression equation with a calculated R 2 = 0.98.

    Article Snippet: Binding Kinetics for Purified Hsp90 and Hsp90 from Cancer Cell Lysates For dissociation off-rate determinations, a [3 H]17-AAG·Hsp90 complex was formed by incubating radiolabeled 17-AAG (200 nm ) with purified Hsp90 (100 nm ) or SK-BR-3 lysates (∼100 nm Hsp90 as determined by quantitative immunoblotting) at 4 °C overnight in Hsp90 binding buffer (20 mm Hepes, pH 7.3, 1 mm EDTA, 100 mm KCl, 5 mm MgCl, 0.01% (v/v) Nonidet P-40, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific), 0.5 mg/ml bovine gamma globulin, and protease inhibitor mixture (Roche Diagnostics GmbH).

    Techniques: Activity Assay, Mouse Assay, Standard Deviation

    Effect of Hsp90 occupancy on client protein abundance. H1650 cells were incubated for 6 or 24 h with increasing concentrations of IPI-504. HER2, mEGFR, Akt, and cRaf protein levels were assessed by immunoblotting ( A and C ). The fraction of degraded proteins was assessed by densitometry compared with untreated samples and plotted together with the occupancy curves ( B and D ).

    Journal: The Journal of Biological Chemistry

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    doi: 10.1074/jbc.M110.141580

    Figure Lengend Snippet: Effect of Hsp90 occupancy on client protein abundance. H1650 cells were incubated for 6 or 24 h with increasing concentrations of IPI-504. HER2, mEGFR, Akt, and cRaf protein levels were assessed by immunoblotting ( A and C ). The fraction of degraded proteins was assessed by densitometry compared with untreated samples and plotted together with the occupancy curves ( B and D ).

    Article Snippet: Binding Kinetics for Purified Hsp90 and Hsp90 from Cancer Cell Lysates For dissociation off-rate determinations, a [3 H]17-AAG·Hsp90 complex was formed by incubating radiolabeled 17-AAG (200 nm ) with purified Hsp90 (100 nm ) or SK-BR-3 lysates (∼100 nm Hsp90 as determined by quantitative immunoblotting) at 4 °C overnight in Hsp90 binding buffer (20 mm Hepes, pH 7.3, 1 mm EDTA, 100 mm KCl, 5 mm MgCl, 0.01% (v/v) Nonidet P-40, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific), 0.5 mg/ml bovine gamma globulin, and protease inhibitor mixture (Roche Diagnostics GmbH).

    Techniques: Incubation

    Binding of [ 3 H]17-AAG to purified Hela Hsp90 and SK-BR-3 lysates at 4 °C. The binding reaction was initiated by adding 10 μ m [ 3 H]17-AAG to 100 n m purified Hsp90 or to SK-BR-3 lysate containing ∼100 n m Hsp90. Drug association was measured at 4 °C by a time-dependent increase in protein bound counts for purified Hsp90 ( A ) or Hsp90 in SK-BR-3 cell lysate ( B ). Data were fitted by nonlinear regression to a single exponential equation to obtain a ( k obs ) value. Half-life was calculated using the equation t ½ = 0.693/ k obs .

    Journal: The Journal of Biological Chemistry

    Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo *

    doi: 10.1074/jbc.M110.141580

    Figure Lengend Snippet: Binding of [ 3 H]17-AAG to purified Hela Hsp90 and SK-BR-3 lysates at 4 °C. The binding reaction was initiated by adding 10 μ m [ 3 H]17-AAG to 100 n m purified Hsp90 or to SK-BR-3 lysate containing ∼100 n m Hsp90. Drug association was measured at 4 °C by a time-dependent increase in protein bound counts for purified Hsp90 ( A ) or Hsp90 in SK-BR-3 cell lysate ( B ). Data were fitted by nonlinear regression to a single exponential equation to obtain a ( k obs ) value. Half-life was calculated using the equation t ½ = 0.693/ k obs .

    Article Snippet: Binding Kinetics for Purified Hsp90 and Hsp90 from Cancer Cell Lysates For dissociation off-rate determinations, a [3 H]17-AAG·Hsp90 complex was formed by incubating radiolabeled 17-AAG (200 nm ) with purified Hsp90 (100 nm ) or SK-BR-3 lysates (∼100 nm Hsp90 as determined by quantitative immunoblotting) at 4 °C overnight in Hsp90 binding buffer (20 mm Hepes, pH 7.3, 1 mm EDTA, 100 mm KCl, 5 mm MgCl, 0.01% (v/v) Nonidet P-40, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific), 0.5 mg/ml bovine gamma globulin, and protease inhibitor mixture (Roche Diagnostics GmbH).

    Techniques: Binding Assay, Purification