Structured Review

Millipore tris
Detergent inhibits shFcRn albumin interaction. <t>Sepharose</t> (S)-HSA, S-IgG and <t>S-Tris</t> (A–C) were incubated with shFcRn in a pH 6.0 buffer containing indicated concentrations of OG as shown. Bound shFcRn was eluted and quantified by immunoblotting with anti-FcRn antibody. The positions of molecular weight markers (M, in kD) are shown. Lane 1 in each case shows the eluate from Sepharose-ligand in the absence of shFcRn. Lane 2 in all the gels contained 10 μg shFcRn, the amount added to every adsorbent sample. The shFcRn bands were quantified and plotted in panel D vs. OG concentration. Similar results were obtained in another experiment.
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Images

1) Product Images from "The Major Histocompatibility Complex-related Fc Receptor for IgG (FcRn) Binds Albumin and Prolongs Its Lifespan"

Article Title: The Major Histocompatibility Complex-related Fc Receptor for IgG (FcRn) Binds Albumin and Prolongs Its Lifespan

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20021829

Detergent inhibits shFcRn albumin interaction. Sepharose (S)-HSA, S-IgG and S-Tris (A–C) were incubated with shFcRn in a pH 6.0 buffer containing indicated concentrations of OG as shown. Bound shFcRn was eluted and quantified by immunoblotting with anti-FcRn antibody. The positions of molecular weight markers (M, in kD) are shown. Lane 1 in each case shows the eluate from Sepharose-ligand in the absence of shFcRn. Lane 2 in all the gels contained 10 μg shFcRn, the amount added to every adsorbent sample. The shFcRn bands were quantified and plotted in panel D vs. OG concentration. Similar results were obtained in another experiment.
Figure Legend Snippet: Detergent inhibits shFcRn albumin interaction. Sepharose (S)-HSA, S-IgG and S-Tris (A–C) were incubated with shFcRn in a pH 6.0 buffer containing indicated concentrations of OG as shown. Bound shFcRn was eluted and quantified by immunoblotting with anti-FcRn antibody. The positions of molecular weight markers (M, in kD) are shown. Lane 1 in each case shows the eluate from Sepharose-ligand in the absence of shFcRn. Lane 2 in all the gels contained 10 μg shFcRn, the amount added to every adsorbent sample. The shFcRn bands were quantified and plotted in panel D vs. OG concentration. Similar results were obtained in another experiment.

Techniques Used: Incubation, Molecular Weight, Concentration Assay

pH-dependent binding of FcRn to immobilized albumin. Sepharose (S)-HSA, S-IgG, S-Tris, S-fish gelatin (see Materials and Methods), or S-RSA were incubated with shFcRn or srFcRn at varying pH as shown. Bound soluble FcRn were eluted and were quantified by immunoblotting with anti-FcRn antibody. Immunoblots of binding of shFcRn to S-HSA, S-IgG, and S-Tris at pH values indicated are shown in A, B, and C, respectively. Immunoblot of binding of srFcRn to S-RSA is shown in E. The positions of molecular weight markers (M, in kD) are shown. Lane 1 in all the gels contained 15 μg soluble FcRn, the amount added to every adsorbent sample. Lane 2 in each case shows the eluate from Sepharose-ligand in the absence of FcRn. The shFcRn bands were quantified and are plotted in panel D vs. pH. Three distinct experiments have given equivalent results. srFcRn band densities are plotted in panel F vs. pH. A second experiment with srFcRn gave equivalent results. G and H show binding of HFE to S-HSA and S-myoglobin. Lanes 1 and 2 show amounts of shFcRn and HFE added (15 μg) to adsorbent samples. Bound protein was eluted and quantified by blotting with anti-b2m antibody.
Figure Legend Snippet: pH-dependent binding of FcRn to immobilized albumin. Sepharose (S)-HSA, S-IgG, S-Tris, S-fish gelatin (see Materials and Methods), or S-RSA were incubated with shFcRn or srFcRn at varying pH as shown. Bound soluble FcRn were eluted and were quantified by immunoblotting with anti-FcRn antibody. Immunoblots of binding of shFcRn to S-HSA, S-IgG, and S-Tris at pH values indicated are shown in A, B, and C, respectively. Immunoblot of binding of srFcRn to S-RSA is shown in E. The positions of molecular weight markers (M, in kD) are shown. Lane 1 in all the gels contained 15 μg soluble FcRn, the amount added to every adsorbent sample. Lane 2 in each case shows the eluate from Sepharose-ligand in the absence of FcRn. The shFcRn bands were quantified and are plotted in panel D vs. pH. Three distinct experiments have given equivalent results. srFcRn band densities are plotted in panel F vs. pH. A second experiment with srFcRn gave equivalent results. G and H show binding of HFE to S-HSA and S-myoglobin. Lanes 1 and 2 show amounts of shFcRn and HFE added (15 μg) to adsorbent samples. Bound protein was eluted and quantified by blotting with anti-b2m antibody.

Techniques Used: Binding Assay, Fluorescence In Situ Hybridization, Incubation, Western Blot, Molecular Weight

2) Product Images from "The Major Histocompatibility Complex-related Fc Receptor for IgG (FcRn) Binds Albumin and Prolongs Its Lifespan"

Article Title: The Major Histocompatibility Complex-related Fc Receptor for IgG (FcRn) Binds Albumin and Prolongs Its Lifespan

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20021829

Detergent inhibits shFcRn albumin interaction. Sepharose (S)-HSA, S-IgG and S-Tris (A–C) were incubated with shFcRn in a pH 6.0 buffer containing indicated concentrations of OG as shown. Bound shFcRn was eluted and quantified by immunoblotting with anti-FcRn antibody. The positions of molecular weight markers (M, in kD) are shown. Lane 1 in each case shows the eluate from Sepharose-ligand in the absence of shFcRn. Lane 2 in all the gels contained 10 μg shFcRn, the amount added to every adsorbent sample. The shFcRn bands were quantified and plotted in panel D vs. OG concentration. Similar results were obtained in another experiment.
Figure Legend Snippet: Detergent inhibits shFcRn albumin interaction. Sepharose (S)-HSA, S-IgG and S-Tris (A–C) were incubated with shFcRn in a pH 6.0 buffer containing indicated concentrations of OG as shown. Bound shFcRn was eluted and quantified by immunoblotting with anti-FcRn antibody. The positions of molecular weight markers (M, in kD) are shown. Lane 1 in each case shows the eluate from Sepharose-ligand in the absence of shFcRn. Lane 2 in all the gels contained 10 μg shFcRn, the amount added to every adsorbent sample. The shFcRn bands were quantified and plotted in panel D vs. OG concentration. Similar results were obtained in another experiment.

Techniques Used: Incubation, Molecular Weight, Concentration Assay

pH-dependent binding of FcRn to immobilized albumin. Sepharose (S)-HSA, S-IgG, S-Tris, S-fish gelatin (see Materials and Methods), or S-RSA were incubated with shFcRn or srFcRn at varying pH as shown. Bound soluble FcRn were eluted and were quantified by immunoblotting with anti-FcRn antibody. Immunoblots of binding of shFcRn to S-HSA, S-IgG, and S-Tris at pH values indicated are shown in A, B, and C, respectively. Immunoblot of binding of srFcRn to S-RSA is shown in E. The positions of molecular weight markers (M, in kD) are shown. Lane 1 in all the gels contained 15 μg soluble FcRn, the amount added to every adsorbent sample. Lane 2 in each case shows the eluate from Sepharose-ligand in the absence of FcRn. The shFcRn bands were quantified and are plotted in panel D vs. pH. Three distinct experiments have given equivalent results. srFcRn band densities are plotted in panel F vs. pH. A second experiment with srFcRn gave equivalent results. G and H show binding of HFE to S-HSA and S-myoglobin. Lanes 1 and 2 show amounts of shFcRn and HFE added (15 μg) to adsorbent samples. Bound protein was eluted and quantified by blotting with anti-b2m antibody.
Figure Legend Snippet: pH-dependent binding of FcRn to immobilized albumin. Sepharose (S)-HSA, S-IgG, S-Tris, S-fish gelatin (see Materials and Methods), or S-RSA were incubated with shFcRn or srFcRn at varying pH as shown. Bound soluble FcRn were eluted and were quantified by immunoblotting with anti-FcRn antibody. Immunoblots of binding of shFcRn to S-HSA, S-IgG, and S-Tris at pH values indicated are shown in A, B, and C, respectively. Immunoblot of binding of srFcRn to S-RSA is shown in E. The positions of molecular weight markers (M, in kD) are shown. Lane 1 in all the gels contained 15 μg soluble FcRn, the amount added to every adsorbent sample. Lane 2 in each case shows the eluate from Sepharose-ligand in the absence of FcRn. The shFcRn bands were quantified and are plotted in panel D vs. pH. Three distinct experiments have given equivalent results. srFcRn band densities are plotted in panel F vs. pH. A second experiment with srFcRn gave equivalent results. G and H show binding of HFE to S-HSA and S-myoglobin. Lanes 1 and 2 show amounts of shFcRn and HFE added (15 μg) to adsorbent samples. Bound protein was eluted and quantified by blotting with anti-b2m antibody.

Techniques Used: Binding Assay, Fluorescence In Situ Hybridization, Incubation, Western Blot, Molecular Weight

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Clone Assay:

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Centrifugation:

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Synthesized:

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Incubation:

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Activity Assay:

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Expressing:

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BIA-KA:

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Modification:

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Transformation Assay:

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Over Expression:

Article Title: A multisubstrate reductase from Plantago major: structure-function in the short chain reductase superfamily
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High Performance Liquid Chromatography:

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Transfection:

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Chromatography:

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Protease Inhibitor:

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Generated:

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other:

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Sequencing:

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Affinity Purification:

Article Title: Study of Plasmodium falciparum DHHC palmitoyl transferases identifies a role for PfDHHC9 in gametocytogenesis) Study of Plasmodium falciparum DHHC palmitoyl transferases identifies a role for PfDHHC9 in gametocytogenesis
Article Snippet: Briefly, 2 mg of each protein sample was incubated with the following chemicals in PBS: 100 µm biotin‐azide (Life Technologies), 1 mm Tris(2‐carboxyethyl)phosphine (TCEP; Sigma‐Aldrich), 100 µm Tris[(1‐benzyl‐1H ‐1,2,3‐triazol‐4‐yl)methyl]amine (TBTA, Sigma‐Aldrich) dissolved in DMSO/tert‐ butanol (20%/80%), and 1 mm CuSO4 (Sigma‐Aldrich). .. Biotinylated proteins were affinity purified by incubating with a 50 µl bed volume of streptavidin‐agarose (Thermo Scientific) for 2 h at room temperature with rotation.

Binding Assay:

Article Title: The Major Histocompatibility Complex-related Fc Receptor for IgG (FcRn) Binds Albumin and Prolongs Its Lifespan
Article Snippet: Sepharose-HSA, hIgG, and Tris were prepared as described above for binding at varying pH. .. Sepharose beads linked to HSA, hIgG, and Tris (20 μl beads equivalent to ∼180 μg linked protein) were washed with 50 mM Tris, 150 mM NaCl, pH 6.0 buffer with 0.1% fish gelatin and varying concentrations of Octyl-β-D-Glucopyranoside (OG; catalog no. O-8001; Sigma-Aldrich), and were then incubated for 2 h at room temperature with 80 μl of shFcRn (final concentration 100 μg/ml) in 50 mM Tris, 150 mM NaCl, 0.1% fish gelatin pH 6.0 buffer of appropriate detergent concentration.

Mouse Assay:

Article Title: Dissociation of the Dimer of the Intrinsically Disordered Domain of RNase Y upon Antibody Binding
Article Snippet: .. The immunoglobulin (IgG2b, λ x) was produced from mice ascitic fluid, purified by Protein A affinity chromatography by Abmart and stored in 50 mM Tris, 100 mM glycine (pH 9), 0.5% ProClin 300 (Sigma-Aldrich, St. Louis, MO). .. The Fab was generated by papain digestion of the antibody at 37°C in 20 mM sodium phosphate (pH 7.4), 274 mM NaCl, 5.4 mM KCl, 1.25 mM EDTA, 1.25 mM 2-mercaptoethanol using a 0.5% papain/antibody ratio (w/w) for 5 h. Undigested IgG and Fc (fragment crystallizable) fragment were removed by HiTrapQ anion exchange chromatography.

Metabolic Labelling:

Article Title: Study of Plasmodium falciparum DHHC palmitoyl transferases identifies a role for PfDHHC9 in gametocytogenesis) Study of Plasmodium falciparum DHHC palmitoyl transferases identifies a role for PfDHHC9 in gametocytogenesis
Article Snippet: Paragraph title: Purification of palmitoylated proteins by metabolic labelling and click chemistry (palmitoyl‐transferase activity assay) ... Briefly, 2 mg of each protein sample was incubated with the following chemicals in PBS: 100 µm biotin‐azide (Life Technologies), 1 mm Tris(2‐carboxyethyl)phosphine (TCEP; Sigma‐Aldrich), 100 µm Tris[(1‐benzyl‐1H ‐1,2,3‐triazol‐4‐yl)methyl]amine (TBTA, Sigma‐Aldrich) dissolved in DMSO/tert‐ butanol (20%/80%), and 1 mm CuSO4 (Sigma‐Aldrich).

Purification:

Article Title: Folding-Upon-Binding and Signal-On Electrochemical DNA Sensor with High Affinity and Specificity
Article Snippet: .. Materials Reagent-grade chemicals, including (top-oligo(ethylene glycol), HS–(CH2 )11 –OEG6 –OH) TOEG6 (from Prochimia, Poland), 6-mercapto-1-hexanol, tris[hydroxymethyl]aminomethane hydrochloride, tris(2-carboxyethyl) phosphine hydrochloride, sulfuric acid, potassium phosphate monobasic, dibasic, ethanol, and sodium chloride (all from Sigma-Aldrich, St. Louis, Missouri, USA) were used without further purification. .. The clamp-switch and the linear probe were obtained from Biosearch Technologies (Novato, USA) and employed without further purification.

Article Title: Study of Plasmodium falciparum DHHC palmitoyl transferases identifies a role for PfDHHC9 in gametocytogenesis) Study of Plasmodium falciparum DHHC palmitoyl transferases identifies a role for PfDHHC9 in gametocytogenesis
Article Snippet: Paragraph title: Purification of palmitoylated proteins by metabolic labelling and click chemistry (palmitoyl‐transferase activity assay) ... Briefly, 2 mg of each protein sample was incubated with the following chemicals in PBS: 100 µm biotin‐azide (Life Technologies), 1 mm Tris(2‐carboxyethyl)phosphine (TCEP; Sigma‐Aldrich), 100 µm Tris[(1‐benzyl‐1H ‐1,2,3‐triazol‐4‐yl)methyl]amine (TBTA, Sigma‐Aldrich) dissolved in DMSO/tert‐ butanol (20%/80%), and 1 mm CuSO4 (Sigma‐Aldrich).

Article Title: A multisubstrate reductase from Plantago major: structure-function in the short chain reductase superfamily
Article Snippet: Paragraph title: Protein expression and purification ... Cells were harvested by centrifugation and resuspended with lysis buffer; 10 mM imidazole, 50 mM tris(hydroxymethyl)aminomethane (Tris, Sigma), pH 8.0, 100 mM NaCl, 1 mM tris(2-carboxyethyl) phosphine (TCEP, Sigma), 0.1% triton X-100 (Sigma) and 10% glycerol.

Article Title: Dissociation of the Dimer of the Intrinsically Disordered Domain of RNase Y upon Antibody Binding
Article Snippet: .. The immunoglobulin (IgG2b, λ x) was produced from mice ascitic fluid, purified by Protein A affinity chromatography by Abmart and stored in 50 mM Tris, 100 mM glycine (pH 9), 0.5% ProClin 300 (Sigma-Aldrich, St. Louis, MO). .. The Fab was generated by papain digestion of the antibody at 37°C in 20 mM sodium phosphate (pH 7.4), 274 mM NaCl, 5.4 mM KCl, 1.25 mM EDTA, 1.25 mM 2-mercaptoethanol using a 0.5% papain/antibody ratio (w/w) for 5 h. Undigested IgG and Fc (fragment crystallizable) fragment were removed by HiTrapQ anion exchange chromatography.

Plasmid Preparation:

Article Title: A multisubstrate reductase from Plantago major: structure-function in the short chain reductase superfamily
Article Snippet: The plasmid was transformed into BL21 (DE3) cells, the cells were grown at 37 °C in lysogeny broth (LB) media supplemented with 50 μg/ml kanamycin until an optical density at 600 nm of 0.5, after which time the temperature was reduced to 22 °C. .. Cells were harvested by centrifugation and resuspended with lysis buffer; 10 mM imidazole, 50 mM tris(hydroxymethyl)aminomethane (Tris, Sigma), pH 8.0, 100 mM NaCl, 1 mM tris(2-carboxyethyl) phosphine (TCEP, Sigma), 0.1% triton X-100 (Sigma) and 10% glycerol.

Affinity Chromatography:

Article Title: Dissociation of the Dimer of the Intrinsically Disordered Domain of RNase Y upon Antibody Binding
Article Snippet: .. The immunoglobulin (IgG2b, λ x) was produced from mice ascitic fluid, purified by Protein A affinity chromatography by Abmart and stored in 50 mM Tris, 100 mM glycine (pH 9), 0.5% ProClin 300 (Sigma-Aldrich, St. Louis, MO). .. The Fab was generated by papain digestion of the antibody at 37°C in 20 mM sodium phosphate (pH 7.4), 274 mM NaCl, 5.4 mM KCl, 1.25 mM EDTA, 1.25 mM 2-mercaptoethanol using a 0.5% papain/antibody ratio (w/w) for 5 h. Undigested IgG and Fc (fragment crystallizable) fragment were removed by HiTrapQ anion exchange chromatography.

Produced:

Article Title: Dissociation of the Dimer of the Intrinsically Disordered Domain of RNase Y upon Antibody Binding
Article Snippet: .. The immunoglobulin (IgG2b, λ x) was produced from mice ascitic fluid, purified by Protein A affinity chromatography by Abmart and stored in 50 mM Tris, 100 mM glycine (pH 9), 0.5% ProClin 300 (Sigma-Aldrich, St. Louis, MO). .. The Fab was generated by papain digestion of the antibody at 37°C in 20 mM sodium phosphate (pH 7.4), 274 mM NaCl, 5.4 mM KCl, 1.25 mM EDTA, 1.25 mM 2-mercaptoethanol using a 0.5% papain/antibody ratio (w/w) for 5 h. Undigested IgG and Fc (fragment crystallizable) fragment were removed by HiTrapQ anion exchange chromatography.

Concentration Assay:

Article Title: The Major Histocompatibility Complex-related Fc Receptor for IgG (FcRn) Binds Albumin and Prolongs Its Lifespan
Article Snippet: .. Sepharose beads linked to HSA, hIgG, and Tris (20 μl beads equivalent to ∼180 μg linked protein) were washed with 50 mM Tris, 150 mM NaCl, pH 6.0 buffer with 0.1% fish gelatin and varying concentrations of Octyl-β-D-Glucopyranoside (OG; catalog no. O-8001; Sigma-Aldrich), and were then incubated for 2 h at room temperature with 80 μl of shFcRn (final concentration 100 μg/ml) in 50 mM Tris, 150 mM NaCl, 0.1% fish gelatin pH 6.0 buffer of appropriate detergent concentration. .. Unbound protein was washed away using 50 mM Tris, 150 mM NaCl, 0.1% fish gelatin pH 6.0 buffer of appropriate detergent concentration.

Article Title: A multisubstrate reductase from Plantago major: structure-function in the short chain reductase superfamily
Article Snippet: Cells were harvested by centrifugation and resuspended with lysis buffer; 10 mM imidazole, 50 mM tris(hydroxymethyl)aminomethane (Tris, Sigma), pH 8.0, 100 mM NaCl, 1 mM tris(2-carboxyethyl) phosphine (TCEP, Sigma), 0.1% triton X-100 (Sigma) and 10% glycerol. .. The imidazole concentration of the lysis buffer was increased to 20 mM to remove contaminants and the protein eluted with lysis buffer supplemented with 300 mM imidazole.

Liquid Chromatography with Mass Spectroscopy:

Article Title: Nano-scale Liquid Chromatography/Mass Spectrometry and On-the-fly Orthogonal Array Optimization for Quantification of Therapeutic Monoclonal Antibodies and the Application in Preclinical Analysis
Article Snippet: LC/MS grade formic acid was from Fluka (Buchs, Switzerland). .. Tris(2-carboxyethyl)phosphine (TCEP), Tris, iodoacetamide (IAA), and phosphate-buffered saline were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Lysis:

Article Title: A multisubstrate reductase from Plantago major: structure-function in the short chain reductase superfamily
Article Snippet: .. Cells were harvested by centrifugation and resuspended with lysis buffer; 10 mM imidazole, 50 mM tris(hydroxymethyl)aminomethane (Tris, Sigma), pH 8.0, 100 mM NaCl, 1 mM tris(2-carboxyethyl) phosphine (TCEP, Sigma), 0.1% triton X-100 (Sigma) and 10% glycerol. .. Cells were disrupted with a Microfluidics Microfluidizer M-110L Fluid Processer and passed through a 5 mL Ni-NTA superflow Qiagen resin column.

Fluorescence In Situ Hybridization:

Article Title: The Major Histocompatibility Complex-related Fc Receptor for IgG (FcRn) Binds Albumin and Prolongs Its Lifespan
Article Snippet: .. Sepharose beads linked to HSA, hIgG, and Tris (20 μl beads equivalent to ∼180 μg linked protein) were washed with 50 mM Tris, 150 mM NaCl, pH 6.0 buffer with 0.1% fish gelatin and varying concentrations of Octyl-β-D-Glucopyranoside (OG; catalog no. O-8001; Sigma-Aldrich), and were then incubated for 2 h at room temperature with 80 μl of shFcRn (final concentration 100 μg/ml) in 50 mM Tris, 150 mM NaCl, 0.1% fish gelatin pH 6.0 buffer of appropriate detergent concentration. .. Unbound protein was washed away using 50 mM Tris, 150 mM NaCl, 0.1% fish gelatin pH 6.0 buffer of appropriate detergent concentration.

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  • 99
    Millipore tris
    The bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 binds specifically to clathrin triskelia; 15 µg of the bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 was incubated with 0.5 ml <t>clathrin-Sepharose</t> in 0.5 ml isolation buffer at 4°C for 2 hr ( A ), and binding was monitored by batch analysis, as described in Methods. Fraction 1 is the flow-through; fractions 2,3,4 are washes with isolation buffer; and fractions 5,6,7 are eluates with 0.5 M <t>Tris</t> (pH 7.0). All samples were analyzed by SDS-PAGE, followed by silver staining. Negative controls were carried out by incubating 15 µg bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 with 0.5 ml underivatized Sepharose ( B ), and by incubating 15 µg E. coli GST protein with 0.5 ml clathrin-Sepharose ( C ).
    Tris, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1271 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore htt shortstop 44q bis tris gel
    Mammalian-derived expanded <t>Htt</t> exon-1 and shortstop proteins form fibrillar structures as detected by transmission electron microscopy. Time course of GST-Htt exon-1 <t>44Q,</t> A , and GST-Htt shortstop 44Q, B , thrombin cleavage reaction as monitored by TEM.
    Htt Shortstop 44q Bis Tris Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Millipore tris glycine sds 4
    Detection of Full-Length ABI3 Protein in the abi3-5 sua-1 Double Mutant. (A) Immunoblot analysis of ABI3 protein. Total protein was extracted from freshly harvested seeds and separated on a <t>Tris-Gly</t> <t>SDS</t> 4 to 12% polyacrylamide gradient gel. The ABI3 protein is identified as a double band of ∼116 kD in L er , sua-1 , and abi3-5 sua-1 . The truncated ABI3 proteins (ΔABI3) produced by abi3-4 , abi3-5 , and abi3-5 sua-1 and the novel splicing variant of ABI3 are ∼70 kD. Asterisk indicates a nonspecific band that is used as loading control. Sizes of the molecular markers (in kilodaltons) are shown next to the blot. (B) Predicted ABI3 protein isoforms. Gray boxes represent the conserved functional motifs of ABI3 (from left to right: A1, B1, B2, and B3). Boxes with diagonal stripes represent erroneous amino acids stretches.
    Tris Glycine Sds 4, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 binds specifically to clathrin triskelia; 15 µg of the bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 was incubated with 0.5 ml clathrin-Sepharose in 0.5 ml isolation buffer at 4°C for 2 hr ( A ), and binding was monitored by batch analysis, as described in Methods. Fraction 1 is the flow-through; fractions 2,3,4 are washes with isolation buffer; and fractions 5,6,7 are eluates with 0.5 M Tris (pH 7.0). All samples were analyzed by SDS-PAGE, followed by silver staining. Negative controls were carried out by incubating 15 µg bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 with 0.5 ml underivatized Sepharose ( B ), and by incubating 15 µg E. coli GST protein with 0.5 ml clathrin-Sepharose ( C ).

    Journal: Journal of neuroscience research

    Article Title: Bacterially Expressed F1-20/AP-3 Assembles Clathrin Into Cages With a Narrow Size Distribution: Implications for the Regulation of Quantal Size During Neurotransmission

    doi: 10.1002/jnr.490410104

    Figure Lengend Snippet: The bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 binds specifically to clathrin triskelia; 15 µg of the bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 was incubated with 0.5 ml clathrin-Sepharose in 0.5 ml isolation buffer at 4°C for 2 hr ( A ), and binding was monitored by batch analysis, as described in Methods. Fraction 1 is the flow-through; fractions 2,3,4 are washes with isolation buffer; and fractions 5,6,7 are eluates with 0.5 M Tris (pH 7.0). All samples were analyzed by SDS-PAGE, followed by silver staining. Negative controls were carried out by incubating 15 µg bacterially expressed 33 kD NH 2 -terminus of F1-20/AP-3 with 0.5 ml underivatized Sepharose ( B ), and by incubating 15 µg E. coli GST protein with 0.5 ml clathrin-Sepharose ( C ).

    Article Snippet: The Sepharose was then eluted three times with 0.5 ml 0.5 M Tris, 0.1 mM PMSF (pH 7.0) at 37°C for 15 min. Flow-through, washes, and eluates were all concentrated using Millipore quick concentrator-10s, and each sample was brought to 20 µl in IX SDS sample buffer.

    Techniques: Incubation, Isolation, Binding Assay, Flow Cytometry, SDS Page, Silver Staining

    Steady-state kinetics of ATP hydrolysis by the optimized catalytic domain. (A) The ATP hydrolysis is inhibited above 4 mM ATP concentration. (B) The hydrolysis of ATP by the enzyme shows a positive cooperativity up to 4 mM ATP concentration. The kinetics of hydrolysis was measured by following phosphate release in 10 mM Tris, pH = 7.6, 150 mM NaCl, and 1 mM Mg +2 at 37°C as described under Materials and Methods . Total protein concentration was 9.6 µg. Error bars correspond to standard deviation of triplicate measurements.

    Journal: PLoS ONE

    Article Title: Identification of Small-Molecule Inhibitors of Yersinia pestis Type III Secretion System YscN ATPase

    doi: 10.1371/journal.pone.0019716

    Figure Lengend Snippet: Steady-state kinetics of ATP hydrolysis by the optimized catalytic domain. (A) The ATP hydrolysis is inhibited above 4 mM ATP concentration. (B) The hydrolysis of ATP by the enzyme shows a positive cooperativity up to 4 mM ATP concentration. The kinetics of hydrolysis was measured by following phosphate release in 10 mM Tris, pH = 7.6, 150 mM NaCl, and 1 mM Mg +2 at 37°C as described under Materials and Methods . Total protein concentration was 9.6 µg. Error bars correspond to standard deviation of triplicate measurements.

    Article Snippet: The protein fractions were buffer exchanged into 10 mM Tris, pH 7.6, 150 mM NaCl, 10% glycerol and concentrated to 0.6–0.9 mg/ml using the Amicon Ultra-15 concentrators (Millipore).

    Techniques: Concentration Assay, Protein Concentration, Standard Deviation

    Mammalian-derived expanded Htt exon-1 and shortstop proteins form fibrillar structures as detected by transmission electron microscopy. Time course of GST-Htt exon-1 44Q, A , and GST-Htt shortstop 44Q, B , thrombin cleavage reaction as monitored by TEM.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Novel Potentially Toxic Oligomers Formed in Vitro from Mammalian-derived Expanded huntingtin Exon-1 Protein *

    doi: 10.1074/jbc.M111.252577

    Figure Lengend Snippet: Mammalian-derived expanded Htt exon-1 and shortstop proteins form fibrillar structures as detected by transmission electron microscopy. Time course of GST-Htt exon-1 44Q, A , and GST-Htt shortstop 44Q, B , thrombin cleavage reaction as monitored by TEM.

    Article Snippet: For Blue native-PAGE/Western blotting analysis, samples prepared in Blue native sample buffer were resolved on a 3–12% (Htt exon-1 44Q) or 4–16% (Htt shortstop 44Q) Bis-Tris gel and transferred to PVDF (Immobilon P, Millipore) using the Invitrogen system.

    Techniques: Derivative Assay, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy

    Expanded Htt exon-1 and shortstop proteins form a variety of oligomeric and fibrillar aggregates as assessed by atomic force microscopy. A , quantification of numbers of oligomers and fibrils per unit area for both Htt exon-1 44Q and Htt shortstop 44Q

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Novel Potentially Toxic Oligomers Formed in Vitro from Mammalian-derived Expanded huntingtin Exon-1 Protein *

    doi: 10.1074/jbc.M111.252577

    Figure Lengend Snippet: Expanded Htt exon-1 and shortstop proteins form a variety of oligomeric and fibrillar aggregates as assessed by atomic force microscopy. A , quantification of numbers of oligomers and fibrils per unit area for both Htt exon-1 44Q and Htt shortstop 44Q

    Article Snippet: For Blue native-PAGE/Western blotting analysis, samples prepared in Blue native sample buffer were resolved on a 3–12% (Htt exon-1 44Q) or 4–16% (Htt shortstop 44Q) Bis-Tris gel and transferred to PVDF (Immobilon P, Millipore) using the Invitrogen system.

    Techniques: Microscopy

    Expanded Htt exon-1 protein generated in mammalian cells forms SDS-stable and native oligomeric complexes. Time course of GST-Htt exon-1 44Q thrombin cleavage reaction resolved by SDS-PAGE, A , or Blue native-PAGE, B , followed by Western blotting. A , an

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Novel Potentially Toxic Oligomers Formed in Vitro from Mammalian-derived Expanded huntingtin Exon-1 Protein *

    doi: 10.1074/jbc.M111.252577

    Figure Lengend Snippet: Expanded Htt exon-1 protein generated in mammalian cells forms SDS-stable and native oligomeric complexes. Time course of GST-Htt exon-1 44Q thrombin cleavage reaction resolved by SDS-PAGE, A , or Blue native-PAGE, B , followed by Western blotting. A , an

    Article Snippet: For Blue native-PAGE/Western blotting analysis, samples prepared in Blue native sample buffer were resolved on a 3–12% (Htt exon-1 44Q) or 4–16% (Htt shortstop 44Q) Bis-Tris gel and transferred to PVDF (Immobilon P, Millipore) using the Invitrogen system.

    Techniques: Generated, SDS Page, Blue Native PAGE, Western Blot

    Htt-derived oligomeric complexes are not detected by SDS- or Blue native-PAGE for expanded Htt shortstop protein. Time course of GST-Htt shortstop 44Q thrombin cleavage reaction resolved by SDS-PAGE, A , or Blue native-PAGE, B , followed by Western blotting.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Novel Potentially Toxic Oligomers Formed in Vitro from Mammalian-derived Expanded huntingtin Exon-1 Protein *

    doi: 10.1074/jbc.M111.252577

    Figure Lengend Snippet: Htt-derived oligomeric complexes are not detected by SDS- or Blue native-PAGE for expanded Htt shortstop protein. Time course of GST-Htt shortstop 44Q thrombin cleavage reaction resolved by SDS-PAGE, A , or Blue native-PAGE, B , followed by Western blotting.

    Article Snippet: For Blue native-PAGE/Western blotting analysis, samples prepared in Blue native sample buffer were resolved on a 3–12% (Htt exon-1 44Q) or 4–16% (Htt shortstop 44Q) Bis-Tris gel and transferred to PVDF (Immobilon P, Millipore) using the Invitrogen system.

    Techniques: Derivative Assay, Blue Native PAGE, SDS Page, Western Blot

    Detection of Full-Length ABI3 Protein in the abi3-5 sua-1 Double Mutant. (A) Immunoblot analysis of ABI3 protein. Total protein was extracted from freshly harvested seeds and separated on a Tris-Gly SDS 4 to 12% polyacrylamide gradient gel. The ABI3 protein is identified as a double band of ∼116 kD in L er , sua-1 , and abi3-5 sua-1 . The truncated ABI3 proteins (ΔABI3) produced by abi3-4 , abi3-5 , and abi3-5 sua-1 and the novel splicing variant of ABI3 are ∼70 kD. Asterisk indicates a nonspecific band that is used as loading control. Sizes of the molecular markers (in kilodaltons) are shown next to the blot. (B) Predicted ABI3 protein isoforms. Gray boxes represent the conserved functional motifs of ABI3 (from left to right: A1, B1, B2, and B3). Boxes with diagonal stripes represent erroneous amino acids stretches.

    Journal: The Plant Cell

    Article Title: The Conserved Splicing Factor SUA Controls Alternative Splicing of the Developmental Regulator ABI3 in Arabidopsis [W] [W] [OA]

    doi: 10.1105/tpc.110.074674

    Figure Lengend Snippet: Detection of Full-Length ABI3 Protein in the abi3-5 sua-1 Double Mutant. (A) Immunoblot analysis of ABI3 protein. Total protein was extracted from freshly harvested seeds and separated on a Tris-Gly SDS 4 to 12% polyacrylamide gradient gel. The ABI3 protein is identified as a double band of ∼116 kD in L er , sua-1 , and abi3-5 sua-1 . The truncated ABI3 proteins (ΔABI3) produced by abi3-4 , abi3-5 , and abi3-5 sua-1 and the novel splicing variant of ABI3 are ∼70 kD. Asterisk indicates a nonspecific band that is used as loading control. Sizes of the molecular markers (in kilodaltons) are shown next to the blot. (B) Predicted ABI3 protein isoforms. Gray boxes represent the conserved functional motifs of ABI3 (from left to right: A1, B1, B2, and B3). Boxes with diagonal stripes represent erroneous amino acids stretches.

    Article Snippet: After separation on a Tris-glycine SDS 4 to 12% polyacrylamide gradient gel (Anamed), according to , the proteins were blotted on a polyvinylidene fluoride membrane (Millipore) through semidry electrotransfer for 75 min at 2.8 mA/cm2 .

    Techniques: Mutagenesis, Produced, Variant Assay, Functional Assay