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Effect of solution pH on the interaction of Purβ with Acta2 ssDNA. (A) The binding of full-length Purβ (1.0 nM) to PE32-bF ssDNA (0.5 nM) was assessed in assay buffer without MgCl 2 at pH ranging from 7.5 to 12.5. Solid-phase Purβ-PE32-bF complexes were detected by ELISA using rabbit anti-mouse Purβ 210–229 as the primary antibody. Nonspecific background absorbance at 405 nm in control wells without any DNA was subtracted from the signal generated in PE32-bF-coated wells. Background corrected A405 values measured at each pH were normalized by dividing by the mean A405 value determined at pH 7.5 (mean ± SD, n = 4). (B) Effect of solution pH on the thermostability of Purβ. The unfolding of full-length Purβ was evaulated by thermal shift assay at a protein concentration of 2.8 μM in 20 mM <t>HEPES,</t> 150 mM <t>NaCl,</t> 10 mM β-mercaptoethanol adjusted to pH 7.5, 10.5, 11.5, or 12.5.
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1) Product Images from "Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and Acta2 Repression by Purine-Rich Element Binding Protein B"

Article Title: Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and Acta2 Repression by Purine-Rich Element Binding Protein B

Journal: Biochemistry

doi: 10.1021/acs.biochem.6b00006

Effect of solution pH on the interaction of Purβ with Acta2 ssDNA. (A) The binding of full-length Purβ (1.0 nM) to PE32-bF ssDNA (0.5 nM) was assessed in assay buffer without MgCl 2 at pH ranging from 7.5 to 12.5. Solid-phase Purβ-PE32-bF complexes were detected by ELISA using rabbit anti-mouse Purβ 210–229 as the primary antibody. Nonspecific background absorbance at 405 nm in control wells without any DNA was subtracted from the signal generated in PE32-bF-coated wells. Background corrected A405 values measured at each pH were normalized by dividing by the mean A405 value determined at pH 7.5 (mean ± SD, n = 4). (B) Effect of solution pH on the thermostability of Purβ. The unfolding of full-length Purβ was evaulated by thermal shift assay at a protein concentration of 2.8 μM in 20 mM HEPES, 150 mM NaCl, 10 mM β-mercaptoethanol adjusted to pH 7.5, 10.5, 11.5, or 12.5.
Figure Legend Snippet: Effect of solution pH on the interaction of Purβ with Acta2 ssDNA. (A) The binding of full-length Purβ (1.0 nM) to PE32-bF ssDNA (0.5 nM) was assessed in assay buffer without MgCl 2 at pH ranging from 7.5 to 12.5. Solid-phase Purβ-PE32-bF complexes were detected by ELISA using rabbit anti-mouse Purβ 210–229 as the primary antibody. Nonspecific background absorbance at 405 nm in control wells without any DNA was subtracted from the signal generated in PE32-bF-coated wells. Background corrected A405 values measured at each pH were normalized by dividing by the mean A405 value determined at pH 7.5 (mean ± SD, n = 4). (B) Effect of solution pH on the thermostability of Purβ. The unfolding of full-length Purβ was evaulated by thermal shift assay at a protein concentration of 2.8 μM in 20 mM HEPES, 150 mM NaCl, 10 mM β-mercaptoethanol adjusted to pH 7.5, 10.5, 11.5, or 12.5.

Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Generated, Thermal Shift Assay, Protein Concentration

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Transduction:

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Cytometry:

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Blocking Assay:

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Enzyme-linked Immunosorbent Assay:

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Incubation:

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Cell Culture:

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Expressing:

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Modification:

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Transformation Assay:

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Derivative Assay:

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Flow Cytometry:

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Infection:

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Article Title: Neuraminidase inhibition contributes to influenza A virus neutralization by anti-hemagglutinin stem antibodies
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Protein Protein Interaction Assay:

Article Title: Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and Acta2 Repression by Purine-Rich Element Binding Protein B
Article Snippet: Paragraph title: Protein-Protein Interaction Assay. ... Briefly, NHis-tagged MSY1 was diluted to 100 nM in coating buffer consisting of 20 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2 -6H2 O pH 7.5 with 5.0 μg/ml BSA and then applied to microtiter wells (Costar® EIA/RIA plate, 96 Well Easy Wash™, Certified High Binding, Corning, Inc.).

Generated:

Article Title: Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and Acta2 Repression by Purine-Rich Element Binding Protein B
Article Snippet: Briefly, NHis-tagged MSY1 was diluted to 100 nM in coating buffer consisting of 20 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2 -6H2 O pH 7.5 with 5.0 μg/ml BSA and then applied to microtiter wells (Costar® EIA/RIA plate, 96 Well Easy Wash™, Certified High Binding, Corning, Inc.). .. To estimate the relative affinity of wild-type vs. mutant Purβ proteins for MSY1, A 405 readings were corrected for background by subtracting the A 405 signal generated in wells coated with BSA only from the MSY1-coated wells at each concentration of Purβ protein tested.

Inverted Microscopy:

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes
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Binding Assay:

Article Title: Dietary manganese promotes staphylococcal infection of the heart
Article Snippet: S. aureus Newman transformed with pOS1 with the superfolder GFP driven by the sarA promoter and sod ribosomal binding site (Newman pOS1-pSarA-sodRBS-sGFP) was grown to mid-logarithmic phase and pre-opsonized with human serum, as done for the neutrophil killing experiments, or incubated in medium only as a negative control. .. Freshly isolated human neutrophils were suspended in RPMI (Corning, Corning NY), 10 mM HEPES (Corning, Corning NY) and 0.05% human serum albumin (SeraCare, Milford, MA) at a concentration of 5 × 106 /mL.

Article Title: Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and Acta2 Repression by Purine-Rich Element Binding Protein B
Article Snippet: .. Briefly, NHis-tagged MSY1 was diluted to 100 nM in coating buffer consisting of 20 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2 -6H2 O pH 7.5 with 5.0 μg/ml BSA and then applied to microtiter wells (Costar® EIA/RIA plate, 96 Well Easy Wash™, Certified High Binding, Corning, Inc.). .. After a 3 h incubation at room temperature, the wells were washed, blocked, and solutions of either wild-type or mutant Purβ protein were applied in binding buffer consisting of 20 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2 -6H2 O pH 7.5 plus 0.2% (w/v) BSA, 0.05% (v/v) Tween 20, and 0.5 mM DTT.

Magnetic Cell Separation:

Article Title: Anti-CD47 Treatment Stimulates Phagocytosis of Glioblastoma by M1 and M2 Polarized Macrophages and Promotes M1 Polarized Macrophages In Vivo
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Fluorescence:

Article Title: Vascular endothelial growth factor pathway promotes osseointegration and CD31hiEMCNhi endothelium expansion in a mouse tibial implant model
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Mutagenesis:

Article Title: Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and Acta2 Repression by Purine-Rich Element Binding Protein B
Article Snippet: Briefly, NHis-tagged MSY1 was diluted to 100 nM in coating buffer consisting of 20 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2 -6H2 O pH 7.5 with 5.0 μg/ml BSA and then applied to microtiter wells (Costar® EIA/RIA plate, 96 Well Easy Wash™, Certified High Binding, Corning, Inc.). .. After a 3 h incubation at room temperature, the wells were washed, blocked, and solutions of either wild-type or mutant Purβ protein were applied in binding buffer consisting of 20 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2 -6H2 O pH 7.5 plus 0.2% (w/v) BSA, 0.05% (v/v) Tween 20, and 0.5 mM DTT.

Isolation:

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes
Article Snippet: Homotypic aggregation assay Splenic CD8+ T cells isolated by negative selection (Miltenyi Biotec; ) were tested for LFA-1 function by assessing PMA-induced homotypic aggregation as previously described ( ). .. Briefly, T cells at a concentration of 5 × 106 cells/mL in complete media supplemented with 1 mM sodium pyruvate, 1% MEM non-essential amino acids, and 25 mM HEPES (in flat-bottomed 96-well plates, Corning) were pretreated for 15 min with anti-CD11a blocking antibody specific for the αL subunit of LFA-1 (10 µg/mL; BD Biosciences).

Article Title: Effect of ambient temperature and intracellular pigmentation on photothermal damage rate kinetics
Article Snippet: Stock solutions of extracellular MPs from a bovine source were obtained by the isolation method of Dontsov et al. To achieve target pigmentations greater than 200 MPs / cell , multiple additions of 200 – 250 MPs / cell aliquots (in 0.5 mL complete medium) were added to wells 1.5–2 h apart. .. In preparation for laser exposures on the second day postseed, wells with optimal MP uptake and overall health (manual inspection of each well) were carefully washed twice with 0.5 mL Hank’s balanced salt solution (HBSS) with 10 mM HEPES at pH 7.4 and without sodium bicarbonate (Corning 20-023-CV) (collectively called the exposure buffer) at room temperature.

Article Title: Human and murine IL2 receptors differentially respond to the human-IL2 component of immunocytokines
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Article Title: Dietary manganese promotes staphylococcal infection of the heart
Article Snippet: .. Freshly isolated human neutrophils were suspended in RPMI (Corning, Corning NY), 10 mM HEPES (Corning, Corning NY) and 0.05% human serum albumin (SeraCare, Milford, MA) at a concentration of 5 × 106 /mL. ..

Article Title: Structure of the 4-1BB/4-1BBL complex and distinct binding and functional properties of utomilumab and urelumab
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Transfection:

Article Title: A Designed Small Molecule Inhibitor of a Non-Coding RNA Sensitizes HER2 Negative Cancers to Herceptin
Article Snippet: The MCF-10A (CRL-10317, ATCC) cells were cultured in DMEM/F12 50/50 with L-glutamine and 15 mM HEPES (Corning), supplemented with 10% FBS (Sigma), 20 ng/mL human epidermal growth factor (Pepro Tech Inc.), 0.5 mg/mL hydrocortisone (Pfaltz & Bauer), 100 ng/mL cholera toxin (Sigma-Aldrich), 10 μ g/mL insulin (Sigma-Aldrich), and 1× Antibiotic-Antimycotic (Corning). .. Cells were transfected for siRNA knockdown experiments with a control siRNA (Control siRNA-A; sc-37007, Santa Cruz Biotechnology), a sphingosine kinase 1 (SK1) siRNA (SphK1 siRNA (h); sc-44114, Santa Cruz Biotechnology), or a Frizzled 5 (FZD5) siRNA (FZD5 Silencer siRNA; 139070, Invitrogen) using the Lipofectamine RNAiMAX reagent, per the manufacturer’s protocol.

Microscopy:

Article Title: Human and murine IL2 receptors differentially respond to the human-IL2 component of immunocytokines
Article Snippet: For splenocyte isolation, spleens were removed from SCID mice immediately after death and placed in RPMI-1640 complete medium, supplemented with 1x MEM non-essential amino acids (Corning Cellgro, Cat.25-025-CI), 10 mM HEPES (Corning Cellgro, Cat.25-060-CI), 1 mM sodium pyruvate (Corning Cellgro, 25-000-CI), and 0.5 mM β−2-mercaptoethanol (Sigma Chemical, M-6250). .. The spleens were dissociated between the frosted ends of two microscope slides, and erythrocytes were lysed by brief hypotonic shock using cold water.

Purification:

Article Title: Vascular endothelial growth factor pathway promotes osseointegration and CD31hiEMCNhi endothelium expansion in a mouse tibial implant model
Article Snippet: The right tibia (n = 4/group) was collected after the surrounding soft tissue was removed., The proximal one-third tibia was crushed in Hanks Balanced Salt Solution (Life Technologies, Carlsbad, California) containing 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, pH 7.2; Corning Life Sciences, Oneonta, New York) and enzymatically digested with 2.5 mg/ml Collagenase A (Roche, Basel, Switzerland) and 1 unit/ml Dispase II (Roche) for 15 minutes at 37°C under gentle agitation. .. After washing, equal numbers of cells per mouse were blocked with Purified Rat Anti-Mouse CD16/CD32 (BD Biosciences, San Jose, California) for 30 minutes on ice, then stained with allophycocyanin (APC)-conjugated EMCN antibody (eBioscience 50-5851-80), phycoerythrin (PE)-conjugated CD31 (eBioscience 12-0311-81), Alexa Fluor-conjugated CD45 (BioLegend 103128; Bio-Legend, San Diego, California), Brilliant Violet 711-conjugated CD146 (BD Biosciences 740827; BD Biosciences), and AP/Cy7-conjugated Ter119 (BioLegend 116223; BioLegend) for 45 minutes on ice.

Article Title: Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and Acta2 Repression by Purine-Rich Element Binding Protein B
Article Snippet: The binding of purified Purβ proteins to purified MSY1 was evaluated by ELISA as previously described . .. Briefly, NHis-tagged MSY1 was diluted to 100 nM in coating buffer consisting of 20 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2 -6H2 O pH 7.5 with 5.0 μg/ml BSA and then applied to microtiter wells (Costar® EIA/RIA plate, 96 Well Easy Wash™, Certified High Binding, Corning, Inc.).

Selection:

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes
Article Snippet: Homotypic aggregation assay Splenic CD8+ T cells isolated by negative selection (Miltenyi Biotec; ) were tested for LFA-1 function by assessing PMA-induced homotypic aggregation as previously described ( ). .. Briefly, T cells at a concentration of 5 × 106 cells/mL in complete media supplemented with 1 mM sodium pyruvate, 1% MEM non-essential amino acids, and 25 mM HEPES (in flat-bottomed 96-well plates, Corning) were pretreated for 15 min with anti-CD11a blocking antibody specific for the αL subunit of LFA-1 (10 µg/mL; BD Biosciences).

Confocal Microscopy:

Article Title: Structure of the 4-1BB/4-1BBL complex and distinct binding and functional properties of utomilumab and urelumab
Article Snippet: Paragraph title: Confocal microscopy ... Jurkat T cells expressing human 4-1BB receptor on PGK250 wpre-α were cultured using RPMI 1640 (Corning™) reconstituted with 10% FBS (JR Scientific), 100 U/mL penicillin (Gibco) 100 µg/mL streptomycin (Gibco), 2 mM L -glutamine (Gibco), 1 mM sodium pyruvate (Corning), 10 mM HEPES (Corning).

Staining:

Article Title: Vascular endothelial growth factor pathway promotes osseointegration and CD31hiEMCNhi endothelium expansion in a mouse tibial implant model
Article Snippet: The right tibia (n = 4/group) was collected after the surrounding soft tissue was removed., The proximal one-third tibia was crushed in Hanks Balanced Salt Solution (Life Technologies, Carlsbad, California) containing 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, pH 7.2; Corning Life Sciences, Oneonta, New York) and enzymatically digested with 2.5 mg/ml Collagenase A (Roche, Basel, Switzerland) and 1 unit/ml Dispase II (Roche) for 15 minutes at 37°C under gentle agitation. .. After washing, equal numbers of cells per mouse were blocked with Purified Rat Anti-Mouse CD16/CD32 (BD Biosciences, San Jose, California) for 30 minutes on ice, then stained with allophycocyanin (APC)-conjugated EMCN antibody (eBioscience 50-5851-80), phycoerythrin (PE)-conjugated CD31 (eBioscience 12-0311-81), Alexa Fluor-conjugated CD45 (BioLegend 103128; Bio-Legend, San Diego, California), Brilliant Violet 711-conjugated CD146 (BD Biosciences 740827; BD Biosciences), and AP/Cy7-conjugated Ter119 (BioLegend 116223; BioLegend) for 45 minutes on ice.

Article Title: Structure of the 4-1BB/4-1BBL complex and distinct binding and functional properties of utomilumab and urelumab
Article Snippet: Jurkat T cells expressing human 4-1BB receptor on PGK250 wpre-α were cultured using RPMI 1640 (Corning™) reconstituted with 10% FBS (JR Scientific), 100 U/mL penicillin (Gibco) 100 µg/mL streptomycin (Gibco), 2 mM L -glutamine (Gibco), 1 mM sodium pyruvate (Corning), 10 mM HEPES (Corning). .. For staining Jurkat cells were freshly isolated from culture and washed with ice cold staining buffer (PBS + 2% FBS + 0.1% NaN3 ).

Phagocytosis Assay:

Article Title: Dietary manganese promotes staphylococcal infection of the heart
Article Snippet: Paragraph title: Phagocytosis assay ... Freshly isolated human neutrophils were suspended in RPMI (Corning, Corning NY), 10 mM HEPES (Corning, Corning NY) and 0.05% human serum albumin (SeraCare, Milford, MA) at a concentration of 5 × 106 /mL.

Chloramphenicol Acetyltransferase Assay:

Article Title: Human and murine IL2 receptors differentially respond to the human-IL2 component of immunocytokines
Article Snippet: .. For splenocyte isolation, spleens were removed from SCID mice immediately after death and placed in RPMI-1640 complete medium, supplemented with 1x MEM non-essential amino acids (Corning Cellgro, Cat.25-025-CI), 10 mM HEPES (Corning Cellgro, Cat.25-060-CI), 1 mM sodium pyruvate (Corning Cellgro, 25-000-CI), and 0.5 mM β−2-mercaptoethanol (Sigma Chemical, M-6250). .. The spleens were dissociated between the frosted ends of two microscope slides, and erythrocytes were lysed by brief hypotonic shock using cold water.

Mouse Assay:

Article Title: Human and murine IL2 receptors differentially respond to the human-IL2 component of immunocytokines
Article Snippet: .. For splenocyte isolation, spleens were removed from SCID mice immediately after death and placed in RPMI-1640 complete medium, supplemented with 1x MEM non-essential amino acids (Corning Cellgro, Cat.25-025-CI), 10 mM HEPES (Corning Cellgro, Cat.25-060-CI), 1 mM sodium pyruvate (Corning Cellgro, 25-000-CI), and 0.5 mM β−2-mercaptoethanol (Sigma Chemical, M-6250). .. The spleens were dissociated between the frosted ends of two microscope slides, and erythrocytes were lysed by brief hypotonic shock using cold water.

Software:

Article Title: Vascular endothelial growth factor pathway promotes osseointegration and CD31hiEMCNhi endothelium expansion in a mouse tibial implant model
Article Snippet: The right tibia (n = 4/group) was collected after the surrounding soft tissue was removed., The proximal one-third tibia was crushed in Hanks Balanced Salt Solution (Life Technologies, Carlsbad, California) containing 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, pH 7.2; Corning Life Sciences, Oneonta, New York) and enzymatically digested with 2.5 mg/ml Collagenase A (Roche, Basel, Switzerland) and 1 unit/ml Dispase II (Roche) for 15 minutes at 37°C under gentle agitation. .. After washing, cells were resuspended in PBS (pH 7.2) with 2 mM EDTA and 1 μg/ml 4–6, diamidino-2-phenylindole (DAPI; live/dead exclusion) for analysis on an LSRII flow cytometer system (BD Biosciences) and analyzed using FlowJo software (TreeStar, Ashland, Oregon).

Negative Control:

Article Title: Dietary manganese promotes staphylococcal infection of the heart
Article Snippet: S. aureus Newman transformed with pOS1 with the superfolder GFP driven by the sarA promoter and sod ribosomal binding site (Newman pOS1-pSarA-sodRBS-sGFP) was grown to mid-logarithmic phase and pre-opsonized with human serum, as done for the neutrophil killing experiments, or incubated in medium only as a negative control. .. Freshly isolated human neutrophils were suspended in RPMI (Corning, Corning NY), 10 mM HEPES (Corning, Corning NY) and 0.05% human serum albumin (SeraCare, Milford, MA) at a concentration of 5 × 106 /mL.

shRNA:

Article Title: Role of Transmembrane Protein 16F in the Incorporation of Phosphatidylserine Into Budding Ebola Virus Virions
Article Snippet: Paragraph title: Parental and Short Hairpin RNA Cell Lines ... Huh7 and Vero E6 cell lines were obtained from the American Type Culture Collection and cultured in Dulbecco modified Eagle medium and modified Eagle medium, respectively, supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific), 1% HEPES (Corning), 1% nonessential amino acids (Sigma-Aldrich), 1% sodium pyruvate (Sigma-Aldrich), and 2% PenStrep mix (Thermo Fisher Scientific).

Concentration Assay:

Article Title: Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes
Article Snippet: .. Briefly, T cells at a concentration of 5 × 106 cells/mL in complete media supplemented with 1 mM sodium pyruvate, 1% MEM non-essential amino acids, and 25 mM HEPES (in flat-bottomed 96-well plates, Corning) were pretreated for 15 min with anti-CD11a blocking antibody specific for the αL subunit of LFA-1 (10 µg/mL; BD Biosciences). .. Cells were then treated with or without PMA (50 ng/mL; Calbiochem) for 18 hr.

Article Title: Dietary manganese promotes staphylococcal infection of the heart
Article Snippet: .. Freshly isolated human neutrophils were suspended in RPMI (Corning, Corning NY), 10 mM HEPES (Corning, Corning NY) and 0.05% human serum albumin (SeraCare, Milford, MA) at a concentration of 5 × 106 /mL. ..

Article Title: Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and Acta2 Repression by Purine-Rich Element Binding Protein B
Article Snippet: Briefly, NHis-tagged MSY1 was diluted to 100 nM in coating buffer consisting of 20 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2 -6H2 O pH 7.5 with 5.0 μg/ml BSA and then applied to microtiter wells (Costar® EIA/RIA plate, 96 Well Easy Wash™, Certified High Binding, Corning, Inc.). .. To estimate the relative affinity of wild-type vs. mutant Purβ proteins for MSY1, A 405 readings were corrected for background by subtracting the A 405 signal generated in wells coated with BSA only from the MSY1-coated wells at each concentration of Purβ protein tested.

Multiple Displacement Amplification:

Article Title: Sex-biased differences in the correlation between epithelial-to-mesenchymal transition-associated genes in cancer cell lines
Article Snippet: MCF7, MDA-MB-231 and Hs578T human breast cancer cell lines were purchased from the Korean Cell Line Bank (KCLB; Korean Cell Line Research Foundation) and cultured as previously described ( ). .. PC-3 and DU-145 human prostate cancer cell lines, and SNU-840 and SK-OV-3 human ovarian cancer cell lines were purchased from the KCLB; Korean Cell Line Research Foundation and cultured in Roswell Park Memorial Institute (RPMI) medium with L-glutamine and 25 mM HEPES, supplemented with 10% fetal bovine serum (Corning Inc.) and 100 µg/ml penicillin-streptomycin.

Article Title: A Designed Small Molecule Inhibitor of a Non-Coding RNA Sensitizes HER2 Negative Cancers to Herceptin
Article Snippet: MDA-MB-231 (HTB-26, ATCC) cells were cultured in RPMI 1640 medium with L-glutamine and 25 mM HEPES (Corning) supplemented with 10% FBS (Sigma) and 1× Antibiotic-Antimycotic (Corning). .. The MCF-10A (CRL-10317, ATCC) cells were cultured in DMEM/F12 50/50 with L-glutamine and 15 mM HEPES (Corning), supplemented with 10% FBS (Sigma), 20 ng/mL human epidermal growth factor (Pepro Tech Inc.), 0.5 mg/mL hydrocortisone (Pfaltz & Bauer), 100 ng/mL cholera toxin (Sigma-Aldrich), 10 μ g/mL insulin (Sigma-Aldrich), and 1× Antibiotic-Antimycotic (Corning).

FACS:

Article Title: Vascular endothelial growth factor pathway promotes osseointegration and CD31hiEMCNhi endothelium expansion in a mouse tibial implant model
Article Snippet: Paragraph title: Fluorescence-activated cell sorting (FACS). ... The right tibia (n = 4/group) was collected after the surrounding soft tissue was removed., The proximal one-third tibia was crushed in Hanks Balanced Salt Solution (Life Technologies, Carlsbad, California) containing 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, pH 7.2; Corning Life Sciences, Oneonta, New York) and enzymatically digested with 2.5 mg/ml Collagenase A (Roche, Basel, Switzerland) and 1 unit/ml Dispase II (Roche) for 15 minutes at 37°C under gentle agitation.

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  • 99
    Corning Life Sciences hepes buffer
    PA 83 cleavage specificity and its potential role in ANTXR1 receptor binding. Hydrolysis of <t>FRET-peptide</t> PEK-054 and its derivatives using 20 µg/mL PA 83 or LF a No hydrolysis: n.h. (A). ANTXR1 sequence alignment was performed using software programs ClustalW and ESPript 3.0 (Risler matrix, global score of 0.7). Sequence overlap between ANTXR1 and the peptide is depicted in red (B). Presence of the KVLP-region on the outside of the ANTXR1 protein is marked yellow. The 3D structure of the protein was generated using the Cn3D 4.3 program (C). Hydrolysis of ANTXR1 peptide (L109-D117) using 20 µg/mL PA 83 , 20 µg/mL LF or <t>HEPES</t> buffer (D). Results are expressed as mean ± SEM ( n = 3).
    Hepes Buffer, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hepes buffer/product/Corning Life Sciences
    Average 99 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    hepes buffer - by Bioz Stars, 2020-03
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    99
    Corning Life Sciences hepes
    Effect of solution pH on the interaction of Purβ with Acta2 ssDNA. (A) The binding of full-length Purβ (1.0 nM) to PE32-bF ssDNA (0.5 nM) was assessed in assay buffer without MgCl 2 at pH ranging from 7.5 to 12.5. Solid-phase Purβ-PE32-bF complexes were detected by ELISA using rabbit anti-mouse Purβ 210–229 as the primary antibody. Nonspecific background absorbance at 405 nm in control wells without any DNA was subtracted from the signal generated in PE32-bF-coated wells. Background corrected A405 values measured at each pH were normalized by dividing by the mean A405 value determined at pH 7.5 (mean ± SD, n = 4). (B) Effect of solution pH on the thermostability of Purβ. The unfolding of full-length Purβ was evaulated by thermal shift assay at a protein concentration of 2.8 μM in 20 mM <t>HEPES,</t> 150 mM <t>NaCl,</t> 10 mM β-mercaptoethanol adjusted to pH 7.5, 10.5, 11.5, or 12.5.
    Hepes, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 99/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hepes/product/Corning Life Sciences
    Average 99 stars, based on 45 article reviews
    Price from $9.99 to $1999.99
    hepes - by Bioz Stars, 2020-03
    99/100 stars
      Buy from Supplier

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    PA 83 cleavage specificity and its potential role in ANTXR1 receptor binding. Hydrolysis of FRET-peptide PEK-054 and its derivatives using 20 µg/mL PA 83 or LF a No hydrolysis: n.h. (A). ANTXR1 sequence alignment was performed using software programs ClustalW and ESPript 3.0 (Risler matrix, global score of 0.7). Sequence overlap between ANTXR1 and the peptide is depicted in red (B). Presence of the KVLP-region on the outside of the ANTXR1 protein is marked yellow. The 3D structure of the protein was generated using the Cn3D 4.3 program (C). Hydrolysis of ANTXR1 peptide (L109-D117) using 20 µg/mL PA 83 , 20 µg/mL LF or HEPES buffer (D). Results are expressed as mean ± SEM ( n = 3).

    Journal: Virulence

    Article Title: Anthrax protective antigen is a calcium-dependent serine protease

    doi: 10.1080/21505594.2018.1486139

    Figure Lengend Snippet: PA 83 cleavage specificity and its potential role in ANTXR1 receptor binding. Hydrolysis of FRET-peptide PEK-054 and its derivatives using 20 µg/mL PA 83 or LF a No hydrolysis: n.h. (A). ANTXR1 sequence alignment was performed using software programs ClustalW and ESPript 3.0 (Risler matrix, global score of 0.7). Sequence overlap between ANTXR1 and the peptide is depicted in red (B). Presence of the KVLP-region on the outside of the ANTXR1 protein is marked yellow. The 3D structure of the protein was generated using the Cn3D 4.3 program (C). Hydrolysis of ANTXR1 peptide (L109-D117) using 20 µg/mL PA 83 , 20 µg/mL LF or HEPES buffer (D). Results are expressed as mean ± SEM ( n = 3).

    Article Snippet: Protease assay Proteolytic activity was determined, as described earlier [ ], by incubating 16 µM FRET-peptide with varying concentrations of PA83 or LF (both List Laboratories) in HEPES buffer (20 mM HEPES, 0.05% Tween-20, pH 8.2) using blackwell clear bottom 96-well plates (Corning).

    Techniques: Binding Assay, Sequencing, Software, Generated

    PA 83 cleavage specificity and its potential role in ANTXR1 receptor binding. Hydrolysis of FRET-peptide PEK-054 and its derivatives using 20 µg/mL PA 83 or LF a No hydrolysis: n.h. (A). ANTXR1 sequence alignment was performed using software programs ClustalW and ESPript 3.0 (Risler matrix, global score of 0.7). Sequence overlap between ANTXR1 and the peptide is depicted in red (B). Presence of the KVLP-region on the outside of the ANTXR1 protein is marked yellow. The 3D structure of the protein was generated using the Cn3D 4.3 program (C). Hydrolysis of ANTXR1 peptide (L109-D117) using 20 µg/mL PA 83 , 20 µg/mL LF or HEPES buffer (D). Results are expressed as mean ± SEM ( n = 3).

    Journal: Virulence

    Article Title: Anthrax protective antigen is a calcium-dependent serine protease

    doi: 10.1080/21505594.2018.1486139

    Figure Lengend Snippet: PA 83 cleavage specificity and its potential role in ANTXR1 receptor binding. Hydrolysis of FRET-peptide PEK-054 and its derivatives using 20 µg/mL PA 83 or LF a No hydrolysis: n.h. (A). ANTXR1 sequence alignment was performed using software programs ClustalW and ESPript 3.0 (Risler matrix, global score of 0.7). Sequence overlap between ANTXR1 and the peptide is depicted in red (B). Presence of the KVLP-region on the outside of the ANTXR1 protein is marked yellow. The 3D structure of the protein was generated using the Cn3D 4.3 program (C). Hydrolysis of ANTXR1 peptide (L109-D117) using 20 µg/mL PA 83 , 20 µg/mL LF or HEPES buffer (D). Results are expressed as mean ± SEM ( n = 3).

    Article Snippet: Proteolytic activity was determined, as described earlier [ ], by incubating 16 µM FRET-peptide with varying concentrations of PA83 or LF (both List Laboratories) in HEPES buffer (20 mM HEPES, 0.05% Tween-20, pH 8.2) using blackwell clear bottom 96-well plates (Corning).

    Techniques: Binding Assay, Sequencing, Software, Generated

    Effect of solution pH on the interaction of Purβ with Acta2 ssDNA. (A) The binding of full-length Purβ (1.0 nM) to PE32-bF ssDNA (0.5 nM) was assessed in assay buffer without MgCl 2 at pH ranging from 7.5 to 12.5. Solid-phase Purβ-PE32-bF complexes were detected by ELISA using rabbit anti-mouse Purβ 210–229 as the primary antibody. Nonspecific background absorbance at 405 nm in control wells without any DNA was subtracted from the signal generated in PE32-bF-coated wells. Background corrected A405 values measured at each pH were normalized by dividing by the mean A405 value determined at pH 7.5 (mean ± SD, n = 4). (B) Effect of solution pH on the thermostability of Purβ. The unfolding of full-length Purβ was evaulated by thermal shift assay at a protein concentration of 2.8 μM in 20 mM HEPES, 150 mM NaCl, 10 mM β-mercaptoethanol adjusted to pH 7.5, 10.5, 11.5, or 12.5.

    Journal: Biochemistry

    Article Title: Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and Acta2 Repression by Purine-Rich Element Binding Protein B

    doi: 10.1021/acs.biochem.6b00006

    Figure Lengend Snippet: Effect of solution pH on the interaction of Purβ with Acta2 ssDNA. (A) The binding of full-length Purβ (1.0 nM) to PE32-bF ssDNA (0.5 nM) was assessed in assay buffer without MgCl 2 at pH ranging from 7.5 to 12.5. Solid-phase Purβ-PE32-bF complexes were detected by ELISA using rabbit anti-mouse Purβ 210–229 as the primary antibody. Nonspecific background absorbance at 405 nm in control wells without any DNA was subtracted from the signal generated in PE32-bF-coated wells. Background corrected A405 values measured at each pH were normalized by dividing by the mean A405 value determined at pH 7.5 (mean ± SD, n = 4). (B) Effect of solution pH on the thermostability of Purβ. The unfolding of full-length Purβ was evaulated by thermal shift assay at a protein concentration of 2.8 μM in 20 mM HEPES, 150 mM NaCl, 10 mM β-mercaptoethanol adjusted to pH 7.5, 10.5, 11.5, or 12.5.

    Article Snippet: Briefly, NHis-tagged MSY1 was diluted to 100 nM in coating buffer consisting of 20 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2 -6H2 O pH 7.5 with 5.0 μg/ml BSA and then applied to microtiter wells (Costar® EIA/RIA plate, 96 Well Easy Wash™, Certified High Binding, Corning, Inc.).

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Generated, Thermal Shift Assay, Protein Concentration