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Roche tris hcl buffer
Pea chloroplast <t>FBPase</t> oxidation under atmospheric conditions. A, western blot analysis of the in vitro oxidation of the recombinant FBPase. Recombinant cFBP1 was incubated in a solution with 100 mM <t>Tris-HCl</t> (pH 8.0) and 50 mM DTT. After a 30 min incubation at room temperature free cysteines were blocked by adding four volumes of an alkylating solution containing 100 mM Tris-HCl (pH 8.0), 2.7% CHAPS, and 40 mM IAM (lane 1) or after removing excess DTT by gel filtration before adding the alkylating solution (lane 2). 100 ng of the recombinant protein were loaded per lane. B, western blot analysis of the in vitro oxidation of the chloroplast FBPase present in pea extracts. Leaves soluble proteins (detailed extraction protocol in the Section 2 ) were incubated with DTT and free cysteines alkylated as described in panel A. 10 μg of total soluble protein were loaded per lane. 1, non-reduced protein extract; 2, DTT-incubated protein extract; 3, DTT-incubated protein extract after gel filtration. C, schematic model representing the pea FBPase oxidation exposed to atmospheric air. The green colour represents reduced subunits and the red colour represents oxidized subunits. The structures were prepared using PyMOL Molecular Graphics software ( www.pymol.org ). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Images

1) Product Images from "Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum"

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum

Journal: Redox Biology

doi: 10.1016/j.redox.2017.10.008

Pea chloroplast FBPase oxidation under atmospheric conditions. A, western blot analysis of the in vitro oxidation of the recombinant FBPase. Recombinant cFBP1 was incubated in a solution with 100 mM Tris-HCl (pH 8.0) and 50 mM DTT. After a 30 min incubation at room temperature free cysteines were blocked by adding four volumes of an alkylating solution containing 100 mM Tris-HCl (pH 8.0), 2.7% CHAPS, and 40 mM IAM (lane 1) or after removing excess DTT by gel filtration before adding the alkylating solution (lane 2). 100 ng of the recombinant protein were loaded per lane. B, western blot analysis of the in vitro oxidation of the chloroplast FBPase present in pea extracts. Leaves soluble proteins (detailed extraction protocol in the Section 2 ) were incubated with DTT and free cysteines alkylated as described in panel A. 10 μg of total soluble protein were loaded per lane. 1, non-reduced protein extract; 2, DTT-incubated protein extract; 3, DTT-incubated protein extract after gel filtration. C, schematic model representing the pea FBPase oxidation exposed to atmospheric air. The green colour represents reduced subunits and the red colour represents oxidized subunits. The structures were prepared using PyMOL Molecular Graphics software ( www.pymol.org ). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Figure Legend Snippet: Pea chloroplast FBPase oxidation under atmospheric conditions. A, western blot analysis of the in vitro oxidation of the recombinant FBPase. Recombinant cFBP1 was incubated in a solution with 100 mM Tris-HCl (pH 8.0) and 50 mM DTT. After a 30 min incubation at room temperature free cysteines were blocked by adding four volumes of an alkylating solution containing 100 mM Tris-HCl (pH 8.0), 2.7% CHAPS, and 40 mM IAM (lane 1) or after removing excess DTT by gel filtration before adding the alkylating solution (lane 2). 100 ng of the recombinant protein were loaded per lane. B, western blot analysis of the in vitro oxidation of the chloroplast FBPase present in pea extracts. Leaves soluble proteins (detailed extraction protocol in the Section 2 ) were incubated with DTT and free cysteines alkylated as described in panel A. 10 μg of total soluble protein were loaded per lane. 1, non-reduced protein extract; 2, DTT-incubated protein extract; 3, DTT-incubated protein extract after gel filtration. C, schematic model representing the pea FBPase oxidation exposed to atmospheric air. The green colour represents reduced subunits and the red colour represents oxidized subunits. The structures were prepared using PyMOL Molecular Graphics software ( www.pymol.org ). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Techniques Used: Western Blot, In Vitro, Recombinant, Incubation, Filtration, Software

FBPase oxidation by GSNO incubation. A, western blot analyses of the FBPase oxidation after dark incubation with 0.1 mM of the oxidizing molecules GSNO, oxidized glutathione (GSSG), or hydrogen peroxide (H 2 O 2 ) (in a solution with 100 mM Tris-HCl pH 8.0). No oxidizing compound was added to the control condition. Samples were taken at the indicated times and immediately incubated for 1 h at 37 °C with one volume of alkylating buffer (100 mM Tris-HCl [pH 8.0], 4% CHAPS, and 60 mM IAM). B, effect of GSNO concentration on FBPase oxidation. cFBP1 was incubated as in A, with the indicated GSNO concentrations for 2 h. 100 ng of recombinant FBPase were loaded per lane in A and B. C, inactivation of the FBPase activity following 30 min incubations with GSNO. Activity assays were carried out as described in the Section 2 .
Figure Legend Snippet: FBPase oxidation by GSNO incubation. A, western blot analyses of the FBPase oxidation after dark incubation with 0.1 mM of the oxidizing molecules GSNO, oxidized glutathione (GSSG), or hydrogen peroxide (H 2 O 2 ) (in a solution with 100 mM Tris-HCl pH 8.0). No oxidizing compound was added to the control condition. Samples were taken at the indicated times and immediately incubated for 1 h at 37 °C with one volume of alkylating buffer (100 mM Tris-HCl [pH 8.0], 4% CHAPS, and 60 mM IAM). B, effect of GSNO concentration on FBPase oxidation. cFBP1 was incubated as in A, with the indicated GSNO concentrations for 2 h. 100 ng of recombinant FBPase were loaded per lane in A and B. C, inactivation of the FBPase activity following 30 min incubations with GSNO. Activity assays were carried out as described in the Section 2 .

Techniques Used: Incubation, Western Blot, Concentration Assay, Recombinant, Activity Assay

2) Product Images from "Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum"

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum

Journal: Redox Biology

doi: 10.1016/j.redox.2017.10.008

Pea chloroplast FBPase oxidation under atmospheric conditions. A, western blot analysis of the in vitro oxidation of the recombinant FBPase. Recombinant cFBP1 was incubated in a solution with 100 mM Tris-HCl (pH 8.0) and 50 mM DTT. After
Figure Legend Snippet: Pea chloroplast FBPase oxidation under atmospheric conditions. A, western blot analysis of the in vitro oxidation of the recombinant FBPase. Recombinant cFBP1 was incubated in a solution with 100 mM Tris-HCl (pH 8.0) and 50 mM DTT. After

Techniques Used: Western Blot, In Vitro, Recombinant, Incubation

3) Product Images from "Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein"

Article Title: Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein

Journal: Journal of Virology

doi: 10.1128/JVI.00999-13

Solution properties of LNYV C-terminal domain. The experiments were performed in a 20 mM Tris-HCl buffer at pH 7.5 containing 150 mM NaCl at 20°C. (A) Molecular mass determined by size-exclusion chromatography combined with detection by multiangle
Figure Legend Snippet: Solution properties of LNYV C-terminal domain. The experiments were performed in a 20 mM Tris-HCl buffer at pH 7.5 containing 150 mM NaCl at 20°C. (A) Molecular mass determined by size-exclusion chromatography combined with detection by multiangle

Techniques Used: Size-exclusion Chromatography

Related Articles

Clone Assay:

Article Title: Structure–function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune‐suppressing activity from recognition
Article Snippet: Heterologous gene expression in E. coli , and protein purification Avr2 lacking the N‐terminal 37 amino acids, corresponding to the protein identified in the xylem sap (Ma et al ., ), was cloned into pOPINS3C (Berrow et al ., ) by In‐Fusion cloning (Clontech, Takara Bio Europe SAS, Saint‐Germain‐en‐Laye, France). .. Bacterial cultures were grown in Luria‐Bertani media to an A 600 0.5–0.8, then protein expression was induced with 1 mM isopropyl‐1‐thio‐β‐d ‐galactopyranoside and incubated at 18°C for 18 h. Following centrifugation, cell pellets were resuspended in 50 mM Tris‐HCl buffer, pH 8.0 containing 0.5 M NaCl, 50 mM glycine, 5% (v/v) glycerol, 20 mM imidazole and protease inhibitors (Complete EDTA‐free tablets, 1 tablet per 50 ml, Roche).

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum
Article Snippet: 2.2 Protein purification, directed mutagenesis, and FBPase activity after GSNO treatments Coding sequences for the mature proteins were amplified by PCR and cloned into the expression vector pET-28b, which adds a His-tag to the N-terminal end of the polypeptide. .. The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

Article Title: Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein
Article Snippet: Paragraph title: Amino acid sequence analysis, cloning, production, and purification of the C-terminal domain of LNYV P. ... The cells were harvested and suspended in 20 mM Tris-HCl buffer at pH 7.5 containing 150 mM NaCl and EDTA-free protease inhibitor cocktail (Roche) (buffer A).

Centrifugation:

Article Title: Structure–function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune‐suppressing activity from recognition
Article Snippet: .. Bacterial cultures were grown in Luria‐Bertani media to an A 600 0.5–0.8, then protein expression was induced with 1 mM isopropyl‐1‐thio‐β‐d ‐galactopyranoside and incubated at 18°C for 18 h. Following centrifugation, cell pellets were resuspended in 50 mM Tris‐HCl buffer, pH 8.0 containing 0.5 M NaCl, 50 mM glycine, 5% (v/v) glycerol, 20 mM imidazole and protease inhibitors (Complete EDTA‐free tablets, 1 tablet per 50 ml, Roche). .. His6 ‐SUMO‐tagged Avr2 was purified from cleared lysate using an ÄKTAxpress system (GE Healthcare, Little Chalfont, UK) by IMAC (Immobilized Metal Affinity Chromatography) on a 5 ml Ni2+ His‐Trap FF column followed by gel filtration on a Superdex 75 26/60 column pre‐equilibrated in 20 mM HEPES, pH 7.5 containing 0.15 M NaCl.

Article Title: High Hydrostatic Pressure Inducible Trimethylamine N-Oxide Reductase Improves the Pressure Tolerance of Piezosensitive Bacteria Vibrio fluvialis
Article Snippet: .. Briefly, QY27 cells of the mid-log phase were collected by centrifugation and re-suspend with Tris-HCl buffer (40 mM, pH 7.4) containing a proper amount of Complete Protease Inhibitor (Roche, Mannheim, Germany). .. Chloroform was added with gentle mixing and then incubated at room temperature for 5 min. A 10-times volume of Tris-HCl buffer was added and mixed by softly rotating the tubes 2–3 times.

Amplification:

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum
Article Snippet: 2.2 Protein purification, directed mutagenesis, and FBPase activity after GSNO treatments Coding sequences for the mature proteins were amplified by PCR and cloned into the expression vector pET-28b, which adds a His-tag to the N-terminal end of the polypeptide. .. The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

Filtration:

Article Title: Structure–function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune‐suppressing activity from recognition
Article Snippet: Bacterial cultures were grown in Luria‐Bertani media to an A 600 0.5–0.8, then protein expression was induced with 1 mM isopropyl‐1‐thio‐β‐d ‐galactopyranoside and incubated at 18°C for 18 h. Following centrifugation, cell pellets were resuspended in 50 mM Tris‐HCl buffer, pH 8.0 containing 0.5 M NaCl, 50 mM glycine, 5% (v/v) glycerol, 20 mM imidazole and protease inhibitors (Complete EDTA‐free tablets, 1 tablet per 50 ml, Roche). .. His6 ‐SUMO‐tagged Avr2 was purified from cleared lysate using an ÄKTAxpress system (GE Healthcare, Little Chalfont, UK) by IMAC (Immobilized Metal Affinity Chromatography) on a 5 ml Ni2+ His‐Trap FF column followed by gel filtration on a Superdex 75 26/60 column pre‐equilibrated in 20 mM HEPES, pH 7.5 containing 0.15 M NaCl.

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum
Article Snippet: Briefly, 2 μg of FBPase were reduced in a solution containing 100 mM Tris-HCl (pH 8.0) and 50 mM DTT for 30 min. Next, the reducing agent was removed by gel filtration through a Micro Bio-Spin 6 column (BioRad) and the enzyme was then incubated in the dark, for 30 min, in a buffer containing 100 mM Tris–HCl (pH 8.0) and the GSNO concentration specified for each experiment. .. The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

Article Title: Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein
Article Snippet: The cells were harvested and suspended in 20 mM Tris-HCl buffer at pH 7.5 containing 150 mM NaCl and EDTA-free protease inhibitor cocktail (Roche) (buffer A). .. The protein was further purified by gel filtration with a Superdex S75 column (GE Healthcare) equilibrated in buffer A.

Mass Spectrometry:

Article Title: ERdj5 Is the ER Reductase that Catalyzes the Removal of Non-Native Disulfides and Correct Folding of the LDL Receptor
Article Snippet: Paragraph title: Mass Spectrometry ... Cells were lysed in 50 mM Tris-HCl buffer (pH 7.4) containing 1% (v/v) Triton X-100, 150 mM NaCl, 2 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) supplemented with protease inhibitor cocktail (Roche).

Stable Transfection:

Article Title: ERdj5 Is the ER Reductase that Catalyzes the Removal of Non-Native Disulfides and Correct Folding of the LDL Receptor
Article Snippet: Mass Spectrometry Confluent HT1080 cells and HT1080 cells stably overexpressing substrate-trapping ERdj5-V5 and the H63Q mutant were rinsed twice with PBS containing 20 mM N -Ethylmaleimide (NEM). .. Cells were lysed in 50 mM Tris-HCl buffer (pH 7.4) containing 1% (v/v) Triton X-100, 150 mM NaCl, 2 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) supplemented with protease inhibitor cocktail (Roche).

Enzyme Activity Assay:

Article Title: ADAM10 Sheddase Activity is a Potential Lung-Cancer Biomarker
Article Snippet: Paragraph title: Metalloprotease enzyme activity assay ... The assay buffer consisted 25 mM Tris-HCl buffer pH 8, supplemented with 0.1 mg/μL BSA, 0.0006% Brij-35, 1% glycerol, 0.5% Roche-tablet (Roche) and 0.5% ThermoFisher Scientific Halt (Halt) broad-spectrum protease inhibitor cocktails without EDTA.

Blocking Assay:

Article Title: Leptin receptor deficiency confers resistance to behavioral effects of fluoxetine and desipramine via separable substrates
Article Snippet: The hippocampus, prefrontal cortex (PFC) and hypothalamus were dissected out on ice and immediately homogenized in a lysis buffer containing 50 mM Tris–HCl buffer, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, phenylmethylsulfonyl fluoride and PhosSTOP Phosphatase Inhibitor Cocktail (Roche Applied Science, Penzberg, Germany). .. Membranes were then incubated with the following primary antibodies diluted in the blocking solution: anti-Akt (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-Akt(Thr308) (1:1000, Cell Signaling), anti-GSK3β (1:1000, Cell Signaling), anti-phospho-GSK3β(Ser9) (1:1000, Cell Signaling), anti-phospho-GSK3β(Ser389) (1:1000, EMD Millipore, Billerica, MA, USA), anti-ERK1/2 (1:1000, Cell Signaling), anti-phospho ERK1/2(Thr202/Tyr204) (1:1000, Cell Signaling), anti-β-actin (1:3000, Cell Signaling), anti-SERT (1:1000, Abcam, Cambridge, MA, USA).

Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease
Article Snippet: The nitrocellulose sheets were then incubated at 4°C overnight in blocking solution with primary polyclonal antibodies against GST (1:5,000; catalog no. G7781, Sigma) and monoclonal antibodies against HAP1 protein (1:300; catalog no. 611302, BD Transduction Laboratories), CacyBP/SIP (1:1,000; catalog no. ab51288, Abcam), or GFP (1:1,000; catalog no. 11814460001, Roche). .. After transfection, they were lysed in 10 mM Tris-HCl buffer (pH 7.5) with 150 mM NaCl, 1% Triton X-100, 1% NP40 (Nonidet P40, nonionic detergent nonylphenoxypolyethoxylethanol), 2 mM EDTA, 0.2 mM phenylmethanesulfonylfluoride (PMSF; serine protease inhibitor), and protease inhibitor cocktail (Roche).

Electrophoresis:

Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease
Article Snippet: After transfection, they were lysed in 10 mM Tris-HCl buffer (pH 7.5) with 150 mM NaCl, 1% Triton X-100, 1% NP40 (Nonidet P40, nonionic detergent nonylphenoxypolyethoxylethanol), 2 mM EDTA, 0.2 mM phenylmethanesulfonylfluoride (PMSF; serine protease inhibitor), and protease inhibitor cocktail (Roche). .. Proteins were resolved by electrophoresis in 8% polyacrylamide gel and transferred to a PVDF membrane, pretreated with methanol and transfer buffer (48 mM Tris, 39 mM glycine and 5% methanol).

Article Title: Both Orai1 and TRPC1 are Involved in Excessive Store-Operated Calcium Entry in Striatal Neurons Expressing Mutant Huntingtin Exon 1
Article Snippet: After transfection, they were lysed in 10 mM Tris-HCl buffer, pH 7.5, with 150 mM NaCl, 1% Triton X-100, 1% NP40 (Nonidet P40, nonionic detergent nonylphenoxypolyethoxylethanol), 2 mM EDTA, 0.2 mM PMSF (serine protease inhibitor, phenylmethanesulfonylfluoride), and protease inhibitor cocktail (Hoffmann–La Roche AG, Germany). .. Proteins were resolved by electrophoresis in 8% polyacrylamide gel and transferred onto nitrocellulose membrane, which was treated with primary polyclonal anti-TRPC1 antibodies (Alomone Labs, Israel) diluted 1: 200 (or anti-Orai1 antibodies (Sigma) diluted 1: 1000) and secondary peroxidase-conjugated goat antibodies to rabbit IgG (Sigma) diluted 1: 30,000.

Article Title: YKL40 in sporadic amyotrophic lateral sclerosis: cerebrospinal fluid levels as a prognosis marker of disease progression
Article Snippet: Frozen samples of frontal cortex area 8 (n=6 sALS, n=6 controls) and anterior horn of the spinal cord at the lumbar level (n=4 sALS, n=4 controls) were homogenized in RIPA lysis buffer composed of 50mM Tris/HCl buffer, pH 7.4 containing 2mM EDTA, 0.2% Nonidet P-40, 1mM PMSF, protease and phosphatase inhibitor cocktail (Roche Molecular Systems, USA). .. Equal amounts of protein (12μg) for each sample were loaded and separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred onto nitrocellulose membranes (Amersham, Freiburg, GE).

Incubation:

Article Title: Leptin receptor deficiency confers resistance to behavioral effects of fluoxetine and desipramine via separable substrates
Article Snippet: The hippocampus, prefrontal cortex (PFC) and hypothalamus were dissected out on ice and immediately homogenized in a lysis buffer containing 50 mM Tris–HCl buffer, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, phenylmethylsulfonyl fluoride and PhosSTOP Phosphatase Inhibitor Cocktail (Roche Applied Science, Penzberg, Germany). .. Membranes were then incubated with the following primary antibodies diluted in the blocking solution: anti-Akt (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-Akt(Thr308) (1:1000, Cell Signaling), anti-GSK3β (1:1000, Cell Signaling), anti-phospho-GSK3β(Ser9) (1:1000, Cell Signaling), anti-phospho-GSK3β(Ser389) (1:1000, EMD Millipore, Billerica, MA, USA), anti-ERK1/2 (1:1000, Cell Signaling), anti-phospho ERK1/2(Thr202/Tyr204) (1:1000, Cell Signaling), anti-β-actin (1:3000, Cell Signaling), anti-SERT (1:1000, Abcam, Cambridge, MA, USA).

Article Title: ERdj5 Is the ER Reductase that Catalyzes the Removal of Non-Native Disulfides and Correct Folding of the LDL Receptor
Article Snippet: Cells were lysed in 50 mM Tris-HCl buffer (pH 7.4) containing 1% (v/v) Triton X-100, 150 mM NaCl, 2 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) supplemented with protease inhibitor cocktail (Roche). .. Clarified lysates were preincubated with protein A sepharose (Generon) before incubation with anti-V5-conjugated agarose beads (Sigma-Aldrich) for 16 hr at 4°C.

Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease
Article Snippet: As a control, secondary polyclonal antibodies against GAPDH (1:1,000; catalog no. sc-25778, Santa Cruz Biotechnology) or vinculin (1:10,000; catalog no. Ab129002, Abcam) and a monoclonal antibody against β-actin (1:10,000; catalog no. A5441, Sigma) were used, followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:10,000; catalog no. A0545, Sigma) for 1 h at room temperature. .. After transfection, they were lysed in 10 mM Tris-HCl buffer (pH 7.5) with 150 mM NaCl, 1% Triton X-100, 1% NP40 (Nonidet P40, nonionic detergent nonylphenoxypolyethoxylethanol), 2 mM EDTA, 0.2 mM phenylmethanesulfonylfluoride (PMSF; serine protease inhibitor), and protease inhibitor cocktail (Roche).

Article Title: Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells
Article Snippet: Protein deglycosylation assay Membrane proteins (200 μg) were deglycosylated in 50 μl of 100 mM Tris-HCl buffer, pH 8.6, with 2 mU of PNGase F (Roche) at 37°C for 12 h. The membranes were reisolated and suspended in 100 mM sodium citrate buffer, pH 6.0, containing 0.1% SDS and 1% NP-40 and 2 mU O -glycanase (Roche). .. After overnight incubation at 37°C, the reaction mixture was mixed with 0.2 volumes of 5× Laemmli's buffer and subjected to SDS-PAGE.

Article Title: Structure–function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune‐suppressing activity from recognition
Article Snippet: .. Bacterial cultures were grown in Luria‐Bertani media to an A 600 0.5–0.8, then protein expression was induced with 1 mM isopropyl‐1‐thio‐β‐d ‐galactopyranoside and incubated at 18°C for 18 h. Following centrifugation, cell pellets were resuspended in 50 mM Tris‐HCl buffer, pH 8.0 containing 0.5 M NaCl, 50 mM glycine, 5% (v/v) glycerol, 20 mM imidazole and protease inhibitors (Complete EDTA‐free tablets, 1 tablet per 50 ml, Roche). .. His6 ‐SUMO‐tagged Avr2 was purified from cleared lysate using an ÄKTAxpress system (GE Healthcare, Little Chalfont, UK) by IMAC (Immobilized Metal Affinity Chromatography) on a 5 ml Ni2+ His‐Trap FF column followed by gel filtration on a Superdex 75 26/60 column pre‐equilibrated in 20 mM HEPES, pH 7.5 containing 0.15 M NaCl.

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum
Article Snippet: Briefly, 2 μg of FBPase were reduced in a solution containing 100 mM Tris-HCl (pH 8.0) and 50 mM DTT for 30 min. Next, the reducing agent was removed by gel filtration through a Micro Bio-Spin 6 column (BioRad) and the enzyme was then incubated in the dark, for 30 min, in a buffer containing 100 mM Tris–HCl (pH 8.0) and the GSNO concentration specified for each experiment. .. The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

Article Title: High Hydrostatic Pressure Inducible Trimethylamine N-Oxide Reductase Improves the Pressure Tolerance of Piezosensitive Bacteria Vibrio fluvialis
Article Snippet: Briefly, QY27 cells of the mid-log phase were collected by centrifugation and re-suspend with Tris-HCl buffer (40 mM, pH 7.4) containing a proper amount of Complete Protease Inhibitor (Roche, Mannheim, Germany). .. Chloroform was added with gentle mixing and then incubated at room temperature for 5 min. A 10-times volume of Tris-HCl buffer was added and mixed by softly rotating the tubes 2–3 times.

Article Title: YKL40 in sporadic amyotrophic lateral sclerosis: cerebrospinal fluid levels as a prognosis marker of disease progression
Article Snippet: Frozen samples of frontal cortex area 8 (n=6 sALS, n=6 controls) and anterior horn of the spinal cord at the lumbar level (n=4 sALS, n=4 controls) were homogenized in RIPA lysis buffer composed of 50mM Tris/HCl buffer, pH 7.4 containing 2mM EDTA, 0.2% Nonidet P-40, 1mM PMSF, protease and phosphatase inhibitor cocktail (Roche Molecular Systems, USA). .. Non-specific bindings were blocked by incubation in 3% albumin in PBS containing 0.2% Tween for 1h at room temperature.

Article Title: A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes
Article Snippet: Lysis buffer contained 50 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl, 5% glycerol, 350 μg/ml benzamidine, 100 mM β-glycerophosphate, 50 mM NaF, 5 mM NaVO3 and complete EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland). .. Lysates were incubated with Triton X-100 (1% final concentration) for 15 min at 4°C.

Activity Assay:

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum
Article Snippet: Paragraph title: Protein purification, directed mutagenesis, and FBPase activity after GSNO treatments ... The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

Expressing:

Article Title: Structure–function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune‐suppressing activity from recognition
Article Snippet: .. Bacterial cultures were grown in Luria‐Bertani media to an A 600 0.5–0.8, then protein expression was induced with 1 mM isopropyl‐1‐thio‐β‐d ‐galactopyranoside and incubated at 18°C for 18 h. Following centrifugation, cell pellets were resuspended in 50 mM Tris‐HCl buffer, pH 8.0 containing 0.5 M NaCl, 50 mM glycine, 5% (v/v) glycerol, 20 mM imidazole and protease inhibitors (Complete EDTA‐free tablets, 1 tablet per 50 ml, Roche). .. His6 ‐SUMO‐tagged Avr2 was purified from cleared lysate using an ÄKTAxpress system (GE Healthcare, Little Chalfont, UK) by IMAC (Immobilized Metal Affinity Chromatography) on a 5 ml Ni2+ His‐Trap FF column followed by gel filtration on a Superdex 75 26/60 column pre‐equilibrated in 20 mM HEPES, pH 7.5 containing 0.15 M NaCl.

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum
Article Snippet: 2.2 Protein purification, directed mutagenesis, and FBPase activity after GSNO treatments Coding sequences for the mature proteins were amplified by PCR and cloned into the expression vector pET-28b, which adds a His-tag to the N-terminal end of the polypeptide. .. The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

Article Title: Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein
Article Snippet: Then, protein expression was induced by adding 1 mM IPTG (isopropyl-β- d -thiogalactopyranoside), and cells were grown for 3 h. Protein with a selenomethionine (SeMet) substitution was produced by growing the bacterial cells in minimal medium and by adding SeMet before induction of the heterologous protein expression. .. The cells were harvested and suspended in 20 mM Tris-HCl buffer at pH 7.5 containing 150 mM NaCl and EDTA-free protease inhibitor cocktail (Roche) (buffer A).

BIA-KA:

Article Title: High Hydrostatic Pressure Inducible Trimethylamine N-Oxide Reductase Improves the Pressure Tolerance of Piezosensitive Bacteria Vibrio fluvialis
Article Snippet: Briefly, QY27 cells of the mid-log phase were collected by centrifugation and re-suspend with Tris-HCl buffer (40 mM, pH 7.4) containing a proper amount of Complete Protease Inhibitor (Roche, Mannheim, Germany). .. The protein quantification was performed with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockland, ME, United States).

Article Title: YKL40 in sporadic amyotrophic lateral sclerosis: cerebrospinal fluid levels as a prognosis marker of disease progression
Article Snippet: Frozen samples of frontal cortex area 8 (n=6 sALS, n=6 controls) and anterior horn of the spinal cord at the lumbar level (n=4 sALS, n=4 controls) were homogenized in RIPA lysis buffer composed of 50mM Tris/HCl buffer, pH 7.4 containing 2mM EDTA, 0.2% Nonidet P-40, 1mM PMSF, protease and phosphatase inhibitor cocktail (Roche Molecular Systems, USA). .. Protein concentration was determined with the BCA method (Thermo Scientific).

Western Blot:

Article Title: Leptin receptor deficiency confers resistance to behavioral effects of fluoxetine and desipramine via separable substrates
Article Snippet: Paragraph title: Western blot assay ... The hippocampus, prefrontal cortex (PFC) and hypothalamus were dissected out on ice and immediately homogenized in a lysis buffer containing 50 mM Tris–HCl buffer, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, phenylmethylsulfonyl fluoride and PhosSTOP Phosphatase Inhibitor Cocktail (Roche Applied Science, Penzberg, Germany).

Article Title: Both Orai1 and TRPC1 are Involved in Excessive Store-Operated Calcium Entry in Striatal Neurons Expressing Mutant Huntingtin Exon 1
Article Snippet: Paragraph title: Protein electrophoresis and western blotting ... After transfection, they were lysed in 10 mM Tris-HCl buffer, pH 7.5, with 150 mM NaCl, 1% Triton X-100, 1% NP40 (Nonidet P40, nonionic detergent nonylphenoxypolyethoxylethanol), 2 mM EDTA, 0.2 mM PMSF (serine protease inhibitor, phenylmethanesulfonylfluoride), and protease inhibitor cocktail (Hoffmann–La Roche AG, Germany).

Derivative Assay:

Article Title: A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes
Article Snippet: Immunoprecipitation experiments Immuprecipitation of Bfa1-GFP was done using a cell pellet derived from a 200 ml cell culture (2 × 107 cells/ml) treated with nocodazole until > 95% of the cells were arrested in metaphase. .. Lysis buffer contained 50 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl, 5% glycerol, 350 μg/ml benzamidine, 100 mM β-glycerophosphate, 50 mM NaF, 5 mM NaVO3 and complete EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland).

Transfection:

Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease
Article Snippet: .. After transfection, they were lysed in 10 mM Tris-HCl buffer (pH 7.5) with 150 mM NaCl, 1% Triton X-100, 1% NP40 (Nonidet P40, nonionic detergent nonylphenoxypolyethoxylethanol), 2 mM EDTA, 0.2 mM phenylmethanesulfonylfluoride (PMSF; serine protease inhibitor), and protease inhibitor cocktail (Roche). .. Proteins were resolved by electrophoresis in 8% polyacrylamide gel and transferred to a PVDF membrane, pretreated with methanol and transfer buffer (48 mM Tris, 39 mM glycine and 5% methanol).

Article Title: Both Orai1 and TRPC1 are Involved in Excessive Store-Operated Calcium Entry in Striatal Neurons Expressing Mutant Huntingtin Exon 1
Article Snippet: .. After transfection, they were lysed in 10 mM Tris-HCl buffer, pH 7.5, with 150 mM NaCl, 1% Triton X-100, 1% NP40 (Nonidet P40, nonionic detergent nonylphenoxypolyethoxylethanol), 2 mM EDTA, 0.2 mM PMSF (serine protease inhibitor, phenylmethanesulfonylfluoride), and protease inhibitor cocktail (Hoffmann–La Roche AG, Germany). .. Proteins were resolved by electrophoresis in 8% polyacrylamide gel and transferred onto nitrocellulose membrane, which was treated with primary polyclonal anti-TRPC1 antibodies (Alomone Labs, Israel) diluted 1: 200 (or anti-Orai1 antibodies (Sigma) diluted 1: 1000) and secondary peroxidase-conjugated goat antibodies to rabbit IgG (Sigma) diluted 1: 30,000.

Chromatography:

Article Title: Structure–function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune‐suppressing activity from recognition
Article Snippet: Bacterial cultures were grown in Luria‐Bertani media to an A 600 0.5–0.8, then protein expression was induced with 1 mM isopropyl‐1‐thio‐β‐d ‐galactopyranoside and incubated at 18°C for 18 h. Following centrifugation, cell pellets were resuspended in 50 mM Tris‐HCl buffer, pH 8.0 containing 0.5 M NaCl, 50 mM glycine, 5% (v/v) glycerol, 20 mM imidazole and protease inhibitors (Complete EDTA‐free tablets, 1 tablet per 50 ml, Roche). .. Avr2 protein was separated from uncleaved protein and His6 ‐SUMO‐tag by IMAC chromatography, then concentrated to 5–7 ml and repurified by gel filtration as above.

Immunoprecipitation:

Article Title: A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes
Article Snippet: Paragraph title: Immunoprecipitation experiments ... Lysis buffer contained 50 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl, 5% glycerol, 350 μg/ml benzamidine, 100 mM β-glycerophosphate, 50 mM NaF, 5 mM NaVO3 and complete EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland).

Protease Inhibitor:

Article Title: A proteogenomic analysis of Shigella flexneri using 2D LC-MALDI TOF/TOF
Article Snippet: .. Sample pre-fractionation procedures Bacterial cells were resuspended in 100 mM Tris-HCl buffer (pH 8.5), containing 7 M Urea, 2 M Thiourea, a protease inhibitor cocktail tablet (Roche Diagnostics, Germany), and Benzonase Nuclease (25 U/ml, Sigma, USA), and then ruptured by ultrasonication. ..

Article Title: ERdj5 Is the ER Reductase that Catalyzes the Removal of Non-Native Disulfides and Correct Folding of the LDL Receptor
Article Snippet: .. Cells were lysed in 50 mM Tris-HCl buffer (pH 7.4) containing 1% (v/v) Triton X-100, 150 mM NaCl, 2 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) supplemented with protease inhibitor cocktail (Roche). .. Clarified lysates were preincubated with protein A sepharose (Generon) before incubation with anti-V5-conjugated agarose beads (Sigma-Aldrich) for 16 hr at 4°C.

Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease
Article Snippet: .. After transfection, they were lysed in 10 mM Tris-HCl buffer (pH 7.5) with 150 mM NaCl, 1% Triton X-100, 1% NP40 (Nonidet P40, nonionic detergent nonylphenoxypolyethoxylethanol), 2 mM EDTA, 0.2 mM phenylmethanesulfonylfluoride (PMSF; serine protease inhibitor), and protease inhibitor cocktail (Roche). .. Proteins were resolved by electrophoresis in 8% polyacrylamide gel and transferred to a PVDF membrane, pretreated with methanol and transfer buffer (48 mM Tris, 39 mM glycine and 5% methanol).

Article Title: Both Orai1 and TRPC1 are Involved in Excessive Store-Operated Calcium Entry in Striatal Neurons Expressing Mutant Huntingtin Exon 1
Article Snippet: .. After transfection, they were lysed in 10 mM Tris-HCl buffer, pH 7.5, with 150 mM NaCl, 1% Triton X-100, 1% NP40 (Nonidet P40, nonionic detergent nonylphenoxypolyethoxylethanol), 2 mM EDTA, 0.2 mM PMSF (serine protease inhibitor, phenylmethanesulfonylfluoride), and protease inhibitor cocktail (Hoffmann–La Roche AG, Germany). .. Proteins were resolved by electrophoresis in 8% polyacrylamide gel and transferred onto nitrocellulose membrane, which was treated with primary polyclonal anti-TRPC1 antibodies (Alomone Labs, Israel) diluted 1: 200 (or anti-Orai1 antibodies (Sigma) diluted 1: 1000) and secondary peroxidase-conjugated goat antibodies to rabbit IgG (Sigma) diluted 1: 30,000.

Article Title: Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein
Article Snippet: .. The cells were harvested and suspended in 20 mM Tris-HCl buffer at pH 7.5 containing 150 mM NaCl and EDTA-free protease inhibitor cocktail (Roche) (buffer A). .. The cells were broken by sonication, and the soluble fraction was loaded onto a Ni2+ column equilibrated in buffer A.

Article Title: High Hydrostatic Pressure Inducible Trimethylamine N-Oxide Reductase Improves the Pressure Tolerance of Piezosensitive Bacteria Vibrio fluvialis
Article Snippet: .. Briefly, QY27 cells of the mid-log phase were collected by centrifugation and re-suspend with Tris-HCl buffer (40 mM, pH 7.4) containing a proper amount of Complete Protease Inhibitor (Roche, Mannheim, Germany). .. Chloroform was added with gentle mixing and then incubated at room temperature for 5 min. A 10-times volume of Tris-HCl buffer was added and mixed by softly rotating the tubes 2–3 times.

Article Title: A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes
Article Snippet: .. Lysis buffer contained 50 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl, 5% glycerol, 350 μg/ml benzamidine, 100 mM β-glycerophosphate, 50 mM NaF, 5 mM NaVO3 and complete EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland). .. Lysates were incubated with Triton X-100 (1% final concentration) for 15 min at 4°C.

Article Title: ADAM10 Sheddase Activity is a Potential Lung-Cancer Biomarker
Article Snippet: .. The assay buffer consisted 25 mM Tris-HCl buffer pH 8, supplemented with 0.1 mg/μL BSA, 0.0006% Brij-35, 1% glycerol, 0.5% Roche-tablet (Roche) and 0.5% ThermoFisher Scientific Halt (Halt) broad-spectrum protease inhibitor cocktails without EDTA. .. The enzymatic reaction was induced by incubating the lysate/substrate solutions in 96-well white-wall, flat-bottom LUMITRAC-200 plates (Sigma Aldrich) at 37o C for 1 to 4 h. Fluorescence was measured in a TECAN-infinite-200-pro fluorimeter (Technical Communities, Inc., San Bruno, CA) using 485 nm excitation and 530 nm emission wavelengths.

Cell Culture:

Article Title: A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes
Article Snippet: Immunoprecipitation experiments Immuprecipitation of Bfa1-GFP was done using a cell pellet derived from a 200 ml cell culture (2 × 107 cells/ml) treated with nocodazole until > 95% of the cells were arrested in metaphase. .. Lysis buffer contained 50 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl, 5% glycerol, 350 μg/ml benzamidine, 100 mM β-glycerophosphate, 50 mM NaF, 5 mM NaVO3 and complete EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland).

Protein Concentration:

Article Title: YKL40 in sporadic amyotrophic lateral sclerosis: cerebrospinal fluid levels as a prognosis marker of disease progression
Article Snippet: Frozen samples of frontal cortex area 8 (n=6 sALS, n=6 controls) and anterior horn of the spinal cord at the lumbar level (n=4 sALS, n=4 controls) were homogenized in RIPA lysis buffer composed of 50mM Tris/HCl buffer, pH 7.4 containing 2mM EDTA, 0.2% Nonidet P-40, 1mM PMSF, protease and phosphatase inhibitor cocktail (Roche Molecular Systems, USA). .. Protein concentration was determined with the BCA method (Thermo Scientific).

Polymerase Chain Reaction:

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum
Article Snippet: 2.2 Protein purification, directed mutagenesis, and FBPase activity after GSNO treatments Coding sequences for the mature proteins were amplified by PCR and cloned into the expression vector pET-28b, which adds a His-tag to the N-terminal end of the polypeptide. .. The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

Sonication:

Article Title: Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein
Article Snippet: The cells were harvested and suspended in 20 mM Tris-HCl buffer at pH 7.5 containing 150 mM NaCl and EDTA-free protease inhibitor cocktail (Roche) (buffer A). .. The cells were broken by sonication, and the soluble fraction was loaded onto a Ni2+ column equilibrated in buffer A.

Recombinant:

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum
Article Snippet: Recombinant proteins were purified by Ni2+ affinity chromatography (GE Healthcare Life Sciences) according to the manufacturer's instructions. .. The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

Article Title: Expression and NMR Structural Studies of Isotopically Labeled Cannabinoid Receptor Type II
Article Snippet: Paragraph title: 5. Purification of Recombinant CB2 Receptor ... Mix the biomass of the cells with 300 mL of ice-cold 50 mM Tris-HCl buffer, pH 7.5 supplemented with Complete, EDTA-free (Roche), 5 mM MgCl2 and DNAse I (Sigma) in a 1L blender.

Article Title: ADAM10 Sheddase Activity is a Potential Lung-Cancer Biomarker
Article Snippet: Metalloprotease enzyme activity assay The assay was performed in 100 μL of assay-buffer solutions of 1 to 10 μg of cell-, tissue- or exosome-lysate proteins, or 10 ng of recombinant ADAM10 (rADAM10) or rADAM17 and supplemented with 10 μM PEPDAB substrates (BioZyme) . .. The assay buffer consisted 25 mM Tris-HCl buffer pH 8, supplemented with 0.1 mg/μL BSA, 0.0006% Brij-35, 1% glycerol, 0.5% Roche-tablet (Roche) and 0.5% ThermoFisher Scientific Halt (Halt) broad-spectrum protease inhibitor cocktails without EDTA.

Nucleic Acid Electrophoresis:

Article Title: Leptin receptor deficiency confers resistance to behavioral effects of fluoxetine and desipramine via separable substrates
Article Snippet: The hippocampus, prefrontal cortex (PFC) and hypothalamus were dissected out on ice and immediately homogenized in a lysis buffer containing 50 mM Tris–HCl buffer, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, phenylmethylsulfonyl fluoride and PhosSTOP Phosphatase Inhibitor Cocktail (Roche Applied Science, Penzberg, Germany). .. The denatured protein was separated on an SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane.

Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease
Article Snippet: Protein extracts (20 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a Protran nitrocellulose membrane (Whatman), and blocked for 2 h at room temperature in TBS-T (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20 plus 5% dry nonfat milk). .. After transfection, they were lysed in 10 mM Tris-HCl buffer (pH 7.5) with 150 mM NaCl, 1% Triton X-100, 1% NP40 (Nonidet P40, nonionic detergent nonylphenoxypolyethoxylethanol), 2 mM EDTA, 0.2 mM phenylmethanesulfonylfluoride (PMSF; serine protease inhibitor), and protease inhibitor cocktail (Roche).

Article Title: YKL40 in sporadic amyotrophic lateral sclerosis: cerebrospinal fluid levels as a prognosis marker of disease progression
Article Snippet: Paragraph title: Gel electrophoresis and immunoblotting ... Frozen samples of frontal cortex area 8 (n=6 sALS, n=6 controls) and anterior horn of the spinal cord at the lumbar level (n=4 sALS, n=4 controls) were homogenized in RIPA lysis buffer composed of 50mM Tris/HCl buffer, pH 7.4 containing 2mM EDTA, 0.2% Nonidet P-40, 1mM PMSF, protease and phosphatase inhibitor cocktail (Roche Molecular Systems, USA).

Protein Electrophoresis:

Article Title: Both Orai1 and TRPC1 are Involved in Excessive Store-Operated Calcium Entry in Striatal Neurons Expressing Mutant Huntingtin Exon 1
Article Snippet: Paragraph title: Protein electrophoresis and western blotting ... After transfection, they were lysed in 10 mM Tris-HCl buffer, pH 7.5, with 150 mM NaCl, 1% Triton X-100, 1% NP40 (Nonidet P40, nonionic detergent nonylphenoxypolyethoxylethanol), 2 mM EDTA, 0.2 mM PMSF (serine protease inhibitor, phenylmethanesulfonylfluoride), and protease inhibitor cocktail (Hoffmann–La Roche AG, Germany).

Fluorescence:

Article Title: ADAM10 Sheddase Activity is a Potential Lung-Cancer Biomarker
Article Snippet: The assay buffer consisted 25 mM Tris-HCl buffer pH 8, supplemented with 0.1 mg/μL BSA, 0.0006% Brij-35, 1% glycerol, 0.5% Roche-tablet (Roche) and 0.5% ThermoFisher Scientific Halt (Halt) broad-spectrum protease inhibitor cocktails without EDTA. .. The enzymatic reaction was induced by incubating the lysate/substrate solutions in 96-well white-wall, flat-bottom LUMITRAC-200 plates (Sigma Aldrich) at 37o C for 1 to 4 h. Fluorescence was measured in a TECAN-infinite-200-pro fluorimeter (Technical Communities, Inc., San Bruno, CA) using 485 nm excitation and 530 nm emission wavelengths.

Mutagenesis:

Article Title: ERdj5 Is the ER Reductase that Catalyzes the Removal of Non-Native Disulfides and Correct Folding of the LDL Receptor
Article Snippet: Mass Spectrometry Confluent HT1080 cells and HT1080 cells stably overexpressing substrate-trapping ERdj5-V5 and the H63Q mutant were rinsed twice with PBS containing 20 mM N -Ethylmaleimide (NEM). .. Cells were lysed in 50 mM Tris-HCl buffer (pH 7.4) containing 1% (v/v) Triton X-100, 150 mM NaCl, 2 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) supplemented with protease inhibitor cocktail (Roche).

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum
Article Snippet: Paragraph title: Protein purification, directed mutagenesis, and FBPase activity after GSNO treatments ... The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

Mouse Assay:

Article Title: Leptin receptor deficiency confers resistance to behavioral effects of fluoxetine and desipramine via separable substrates
Article Snippet: Western blot assay Mice were killed by rapid decapitation. .. The hippocampus, prefrontal cortex (PFC) and hypothalamus were dissected out on ice and immediately homogenized in a lysis buffer containing 50 mM Tris–HCl buffer, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, phenylmethylsulfonyl fluoride and PhosSTOP Phosphatase Inhibitor Cocktail (Roche Applied Science, Penzberg, Germany).

Protein Purification:

Article Title: Structure–function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune‐suppressing activity from recognition
Article Snippet: Paragraph title: Heterologous gene expression in E. coli , and protein purification ... Bacterial cultures were grown in Luria‐Bertani media to an A 600 0.5–0.8, then protein expression was induced with 1 mM isopropyl‐1‐thio‐β‐d ‐galactopyranoside and incubated at 18°C for 18 h. Following centrifugation, cell pellets were resuspended in 50 mM Tris‐HCl buffer, pH 8.0 containing 0.5 M NaCl, 50 mM glycine, 5% (v/v) glycerol, 20 mM imidazole and protease inhibitors (Complete EDTA‐free tablets, 1 tablet per 50 ml, Roche).

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum
Article Snippet: Paragraph title: Protein purification, directed mutagenesis, and FBPase activity after GSNO treatments ... The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

Sequencing:

Article Title: Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein
Article Snippet: Paragraph title: Amino acid sequence analysis, cloning, production, and purification of the C-terminal domain of LNYV P. ... The cells were harvested and suspended in 20 mM Tris-HCl buffer at pH 7.5 containing 150 mM NaCl and EDTA-free protease inhibitor cocktail (Roche) (buffer A).

Affinity Chromatography:

Article Title: Structure–function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune‐suppressing activity from recognition
Article Snippet: Bacterial cultures were grown in Luria‐Bertani media to an A 600 0.5–0.8, then protein expression was induced with 1 mM isopropyl‐1‐thio‐β‐d ‐galactopyranoside and incubated at 18°C for 18 h. Following centrifugation, cell pellets were resuspended in 50 mM Tris‐HCl buffer, pH 8.0 containing 0.5 M NaCl, 50 mM glycine, 5% (v/v) glycerol, 20 mM imidazole and protease inhibitors (Complete EDTA‐free tablets, 1 tablet per 50 ml, Roche). .. His6 ‐SUMO‐tagged Avr2 was purified from cleared lysate using an ÄKTAxpress system (GE Healthcare, Little Chalfont, UK) by IMAC (Immobilized Metal Affinity Chromatography) on a 5 ml Ni2+ His‐Trap FF column followed by gel filtration on a Superdex 75 26/60 column pre‐equilibrated in 20 mM HEPES, pH 7.5 containing 0.15 M NaCl.

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum
Article Snippet: Recombinant proteins were purified by Ni2+ affinity chromatography (GE Healthcare Life Sciences) according to the manufacturer's instructions. .. The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

Construct:

Article Title: Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein
Article Snippet: On the basis of this prediction, a synthetic gene (GeneArt) optimized for expression in Escherichia coli and encoding residues 230 to 300 of LNYV P (71 residues) was cloned between the NcoI and BamHI restriction sites of a pET28 vector, such that the construct includes two additional N-terminal residues (Met-Ala) and a C-terminal His6 tag with a Leu-Glu linker. .. The cells were harvested and suspended in 20 mM Tris-HCl buffer at pH 7.5 containing 150 mM NaCl and EDTA-free protease inhibitor cocktail (Roche) (buffer A).

Polyacrylamide Gel Electrophoresis:

Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease
Article Snippet: Protein extracts (20 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a Protran nitrocellulose membrane (Whatman), and blocked for 2 h at room temperature in TBS-T (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20 plus 5% dry nonfat milk). .. After transfection, they were lysed in 10 mM Tris-HCl buffer (pH 7.5) with 150 mM NaCl, 1% Triton X-100, 1% NP40 (Nonidet P40, nonionic detergent nonylphenoxypolyethoxylethanol), 2 mM EDTA, 0.2 mM phenylmethanesulfonylfluoride (PMSF; serine protease inhibitor), and protease inhibitor cocktail (Roche).

Article Title: YKL40 in sporadic amyotrophic lateral sclerosis: cerebrospinal fluid levels as a prognosis marker of disease progression
Article Snippet: Frozen samples of frontal cortex area 8 (n=6 sALS, n=6 controls) and anterior horn of the spinal cord at the lumbar level (n=4 sALS, n=4 controls) were homogenized in RIPA lysis buffer composed of 50mM Tris/HCl buffer, pH 7.4 containing 2mM EDTA, 0.2% Nonidet P-40, 1mM PMSF, protease and phosphatase inhibitor cocktail (Roche Molecular Systems, USA). .. Equal amounts of protein (12μg) for each sample were loaded and separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred onto nitrocellulose membranes (Amersham, Freiburg, GE).

Purification:

Article Title: Structure–function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune‐suppressing activity from recognition
Article Snippet: Bacterial cultures were grown in Luria‐Bertani media to an A 600 0.5–0.8, then protein expression was induced with 1 mM isopropyl‐1‐thio‐β‐d ‐galactopyranoside and incubated at 18°C for 18 h. Following centrifugation, cell pellets were resuspended in 50 mM Tris‐HCl buffer, pH 8.0 containing 0.5 M NaCl, 50 mM glycine, 5% (v/v) glycerol, 20 mM imidazole and protease inhibitors (Complete EDTA‐free tablets, 1 tablet per 50 ml, Roche). .. His6 ‐SUMO‐tagged Avr2 was purified from cleared lysate using an ÄKTAxpress system (GE Healthcare, Little Chalfont, UK) by IMAC (Immobilized Metal Affinity Chromatography) on a 5 ml Ni2+ His‐Trap FF column followed by gel filtration on a Superdex 75 26/60 column pre‐equilibrated in 20 mM HEPES, pH 7.5 containing 0.15 M NaCl.

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum
Article Snippet: Recombinant proteins were purified by Ni2+ affinity chromatography (GE Healthcare Life Sciences) according to the manufacturer's instructions. .. The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

Article Title: Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein
Article Snippet: Paragraph title: Amino acid sequence analysis, cloning, production, and purification of the C-terminal domain of LNYV P. ... The cells were harvested and suspended in 20 mM Tris-HCl buffer at pH 7.5 containing 150 mM NaCl and EDTA-free protease inhibitor cocktail (Roche) (buffer A).

Article Title: Expression and NMR Structural Studies of Isotopically Labeled Cannabinoid Receptor Type II
Article Snippet: Paragraph title: 5. Purification of Recombinant CB2 Receptor ... Mix the biomass of the cells with 300 mL of ice-cold 50 mM Tris-HCl buffer, pH 7.5 supplemented with Complete, EDTA-free (Roche), 5 mM MgCl2 and DNAse I (Sigma) in a 1L blender.

SDS Page:

Article Title: Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells
Article Snippet: Protein deglycosylation assay Membrane proteins (200 μg) were deglycosylated in 50 μl of 100 mM Tris-HCl buffer, pH 8.6, with 2 mU of PNGase F (Roche) at 37°C for 12 h. The membranes were reisolated and suspended in 100 mM sodium citrate buffer, pH 6.0, containing 0.1% SDS and 1% NP-40 and 2 mU O -glycanase (Roche). .. After overnight incubation at 37°C, the reaction mixture was mixed with 0.2 volumes of 5× Laemmli's buffer and subjected to SDS-PAGE.

Article Title: Structure–function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune‐suppressing activity from recognition
Article Snippet: Bacterial cultures were grown in Luria‐Bertani media to an A 600 0.5–0.8, then protein expression was induced with 1 mM isopropyl‐1‐thio‐β‐d ‐galactopyranoside and incubated at 18°C for 18 h. Following centrifugation, cell pellets were resuspended in 50 mM Tris‐HCl buffer, pH 8.0 containing 0.5 M NaCl, 50 mM glycine, 5% (v/v) glycerol, 20 mM imidazole and protease inhibitors (Complete EDTA‐free tablets, 1 tablet per 50 ml, Roche). .. Fractions containing Avr2 protein, as judged by SDS‐PAGE, were incubated overnight at 4°C with 3C protease (10 μg mg−1 protein) to remove the His6 ‐SUMO‐tag.

Article Title: YKL40 in sporadic amyotrophic lateral sclerosis: cerebrospinal fluid levels as a prognosis marker of disease progression
Article Snippet: Frozen samples of frontal cortex area 8 (n=6 sALS, n=6 controls) and anterior horn of the spinal cord at the lumbar level (n=4 sALS, n=4 controls) were homogenized in RIPA lysis buffer composed of 50mM Tris/HCl buffer, pH 7.4 containing 2mM EDTA, 0.2% Nonidet P-40, 1mM PMSF, protease and phosphatase inhibitor cocktail (Roche Molecular Systems, USA). .. Equal amounts of protein (12μg) for each sample were loaded and separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred onto nitrocellulose membranes (Amersham, Freiburg, GE).

Plasmid Preparation:

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum
Article Snippet: 2.2 Protein purification, directed mutagenesis, and FBPase activity after GSNO treatments Coding sequences for the mature proteins were amplified by PCR and cloned into the expression vector pET-28b, which adds a His-tag to the N-terminal end of the polypeptide. .. The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

Article Title: Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein
Article Snippet: On the basis of this prediction, a synthetic gene (GeneArt) optimized for expression in Escherichia coli and encoding residues 230 to 300 of LNYV P (71 residues) was cloned between the NcoI and BamHI restriction sites of a pET28 vector, such that the construct includes two additional N-terminal residues (Met-Ala) and a C-terminal His6 tag with a Leu-Glu linker. .. The cells were harvested and suspended in 20 mM Tris-HCl buffer at pH 7.5 containing 150 mM NaCl and EDTA-free protease inhibitor cocktail (Roche) (buffer A).

Positron Emission Tomography:

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum
Article Snippet: 2.2 Protein purification, directed mutagenesis, and FBPase activity after GSNO treatments Coding sequences for the mature proteins were amplified by PCR and cloned into the expression vector pET-28b, which adds a His-tag to the N-terminal end of the polypeptide. .. The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

Produced:

Article Title: Structure–function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune‐suppressing activity from recognition
Article Snippet: Avr2 was produced in E. coli SHuffle cells (NEB) (Bessette et al ., ). .. Bacterial cultures were grown in Luria‐Bertani media to an A 600 0.5–0.8, then protein expression was induced with 1 mM isopropyl‐1‐thio‐β‐d ‐galactopyranoside and incubated at 18°C for 18 h. Following centrifugation, cell pellets were resuspended in 50 mM Tris‐HCl buffer, pH 8.0 containing 0.5 M NaCl, 50 mM glycine, 5% (v/v) glycerol, 20 mM imidazole and protease inhibitors (Complete EDTA‐free tablets, 1 tablet per 50 ml, Roche).

Article Title: Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein
Article Snippet: Then, protein expression was induced by adding 1 mM IPTG (isopropyl-β- d -thiogalactopyranoside), and cells were grown for 3 h. Protein with a selenomethionine (SeMet) substitution was produced by growing the bacterial cells in minimal medium and by adding SeMet before induction of the heterologous protein expression. .. The cells were harvested and suspended in 20 mM Tris-HCl buffer at pH 7.5 containing 150 mM NaCl and EDTA-free protease inhibitor cocktail (Roche) (buffer A).

Concentration Assay:

Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum
Article Snippet: Briefly, 2 μg of FBPase were reduced in a solution containing 100 mM Tris-HCl (pH 8.0) and 50 mM DTT for 30 min. Next, the reducing agent was removed by gel filtration through a Micro Bio-Spin 6 column (BioRad) and the enzyme was then incubated in the dark, for 30 min, in a buffer containing 100 mM Tris–HCl (pH 8.0) and the GSNO concentration specified for each experiment. .. The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

Article Title: Expression and NMR Structural Studies of Isotopically Labeled Cannabinoid Receptor Type II
Article Snippet: Mix the biomass of the cells with 300 mL of ice-cold 50 mM Tris-HCl buffer, pH 7.5 supplemented with Complete, EDTA-free (Roche), 5 mM MgCl2 and DNAse I (Sigma) in a 1L blender. .. Add 12x-concentrated stock solution of CHAPS/CHS and 10% DDM so that the final concentration of the detergents in 1L solution will be: 0.5% CHAPS, 0.1% CHS, 1% DDM.

Article Title: A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes
Article Snippet: Lysis buffer contained 50 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl, 5% glycerol, 350 μg/ml benzamidine, 100 mM β-glycerophosphate, 50 mM NaF, 5 mM NaVO3 and complete EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland). .. Lysates were incubated with Triton X-100 (1% final concentration) for 15 min at 4°C.

Lysis:

Article Title: Leptin receptor deficiency confers resistance to behavioral effects of fluoxetine and desipramine via separable substrates
Article Snippet: .. The hippocampus, prefrontal cortex (PFC) and hypothalamus were dissected out on ice and immediately homogenized in a lysis buffer containing 50 mM Tris–HCl buffer, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, phenylmethylsulfonyl fluoride and PhosSTOP Phosphatase Inhibitor Cocktail (Roche Applied Science, Penzberg, Germany). .. The denatured protein was separated on an SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane.

Article Title: ERdj5 Is the ER Reductase that Catalyzes the Removal of Non-Native Disulfides and Correct Folding of the LDL Receptor
Article Snippet: Cells were lysed in 50 mM Tris-HCl buffer (pH 7.4) containing 1% (v/v) Triton X-100, 150 mM NaCl, 2 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) supplemented with protease inhibitor cocktail (Roche). .. Immunoisolates were washed three times with lysis buffer before incubation with 10 mM DTT for 5 min.

Article Title: YKL40 in sporadic amyotrophic lateral sclerosis: cerebrospinal fluid levels as a prognosis marker of disease progression
Article Snippet: .. Frozen samples of frontal cortex area 8 (n=6 sALS, n=6 controls) and anterior horn of the spinal cord at the lumbar level (n=4 sALS, n=4 controls) were homogenized in RIPA lysis buffer composed of 50mM Tris/HCl buffer, pH 7.4 containing 2mM EDTA, 0.2% Nonidet P-40, 1mM PMSF, protease and phosphatase inhibitor cocktail (Roche Molecular Systems, USA). .. Protein concentration was determined with the BCA method (Thermo Scientific).

Article Title: A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes
Article Snippet: .. Lysis buffer contained 50 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl, 5% glycerol, 350 μg/ml benzamidine, 100 mM β-glycerophosphate, 50 mM NaF, 5 mM NaVO3 and complete EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland). .. Lysates were incubated with Triton X-100 (1% final concentration) for 15 min at 4°C.

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  • 99
    Roche tnte buffer
    Mib1 E3 Ligase Interacts with and Destabilizes GABARAP and Promotes GABARAP Ubiquitination at Lys13 and Lys23 (A) HEK293A cells expressing FLAG-Mib1 or control vector for 48 hr were analyzed by immunoblot. (B) Quantification of (A). Statistical analysis using unpaired Student’s t test; mean ± SEM; n = 3; ∗ p ≤ 0.001. (C) Anti-GABARAP immunoprecipitate from HEK293A cells analyzed by immunoblotting. Ab, anti-GABARAP antibody. Lys, HEK293A lysate. (D) HEK293A cells expressing FLAG-tagged constructs were incubated with recombinant GST or GST-GABARAP beads and immunoblotted. (E) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were stringently washed in denaturing buffer. CS, C985S; GAB, GABARAP; Ponc, Ponceau S. Short and long exposures are shown. ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-GABARAP, respectively. (F) Immunoprecipitation of U2OS cells expressing the indicated constructs lysed in boiling SDS buffer and immunoblot. Free ubiquitin and ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-LC3B/GABARAP are indicated, respectively. (G) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were washed as in (E). (H) See (G). Low and high exposures are shown. (I) Immunoprecipitation of HEK293A cells expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in <t>TNTE</t> buffer (20 mM <t>Tris,</t> pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 5 mM EDTA) + N-ethylmaleimide. Diubiquitinated GABARAP is indicated with ∗∗ . Immunoglobulin light chain is indicated with an arrow. (J) Immunoprecipitation of HEK293A cells treated with RF or GABARAP siRNA for 72 hr and expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer + N-ethylmaleimide. Di- and tri-ubiquitinated GABARAP is indicated with ∗∗ and ∗∗∗ , respectively. Immunoglobulin light chain is indicated with an arrow. (K) Two GABARAP ubiquitination sites, lysine 13 (K13) and lysine 23 (K23), were identified by mass spectrometry on three different peptides. (Top) Comparison of peak areas for the FVYKEEHPFEK(diGly)R peptide containing K13 ubiquitination site (n = 3 measurements) is shown. (Middle and bottom) The K23 ubiquitination site was detected as two different peptides as a result of missed cleavage. Quantification of peptides K(diGly)KYPDRVPVIVEK (middle) and K(diGly)KYPDR (bottom) showed significantly lower abundance in C985S mutant compared to the WT. (L) Conservation of GABARAP K13 and K23 ( ∗ ) between ATG8 orthologs. See also Figure S5 .
    Tnte Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche ripa buffer
    Sustained activation of N211Q DDR1b. COS1 cells expressing WT or N211Q DDR1b were serum-starved (18 h) before stimulation (2 h) with (+) 10 μg/ml rat tail collagen I ( Col. I ) or vehicle control (−), as described under “Experimental Procedures.” After stimulation, the media were aspirated, and the cells were washed thoroughly with warm <t>PBS.</t> The dishes were then supplemented with serum-free media and incubated at 37 °C for the indicated times. The cells were lysed with <t>RIPA</t> buffer, and the lysates were analyzed for receptor activation ( A ) and total receptor expression ( B ), as described in Fig. 2 . Black arrow in A indicates phosphorylated DDR1b, and white arrow in B indicates total DDR1b. Anti-Tyr(P) (α- pTyr ).
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    Roche tris hcl buffer
    Pea chloroplast <t>FBPase</t> oxidation under atmospheric conditions. A, western blot analysis of the in vitro oxidation of the recombinant FBPase. Recombinant cFBP1 was incubated in a solution with 100 mM <t>Tris-HCl</t> (pH 8.0) and 50 mM DTT. After a 30 min incubation at room temperature free cysteines were blocked by adding four volumes of an alkylating solution containing 100 mM Tris-HCl (pH 8.0), 2.7% CHAPS, and 40 mM IAM (lane 1) or after removing excess DTT by gel filtration before adding the alkylating solution (lane 2). 100 ng of the recombinant protein were loaded per lane. B, western blot analysis of the in vitro oxidation of the chloroplast FBPase present in pea extracts. Leaves soluble proteins (detailed extraction protocol in the Section 2 ) were incubated with DTT and free cysteines alkylated as described in panel A. 10 μg of total soluble protein were loaded per lane. 1, non-reduced protein extract; 2, DTT-incubated protein extract; 3, DTT-incubated protein extract after gel filtration. C, schematic model representing the pea FBPase oxidation exposed to atmospheric air. The green colour represents reduced subunits and the red colour represents oxidized subunits. The structures were prepared using PyMOL Molecular Graphics software ( www.pymol.org ). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
    Tris Hcl Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche immunoprecipitation buffer
    <t>Co-immunoprecipitation</t> of Htt with SNX21 requires negatively charged residues in the SNX21 N-terminus, but does not require SNX21 to be endosomally localised. (A) Adaptation of SNX20 and SNX21 protein alignment previously generated by Clairfeuille and colleagues ( Clairfeuille et al., 2015 ) . Green boxed regions represent clusters of negatively charged amino acids not present in the SNX20 N-terminal extension. Red boxes denote conserved amino acids within SNX20 and SNX21 sequences and asterisks count every ten residues starting with the fist methionine of SNX20. (B) HEK293-T cells were transiently transfected to express GFP, GFP-tagged full-length SNX21 and two truncation mutants representing the two halves of the N-terminal region of SNX21. Precipitates from the GFP-nanotrap-isolated variants were analysed by western blotting and demonstrate the necessity for a full N-terminal extension in order to facilitate Htt binding. Data are representative of three biological replicates. (C) Site-directed mutagenesis was used to engineer a variety of charge swap mutants targeting the negatively charged clusters of amino acids, prior to probing for Htt binding as above. Two aspartic acid residues in the first N-terminal cluster are essential for precipitation of Htt with SNX21. (D) Both the point mutated GFP-SNX21 and truncation variants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Except for the N-terminal 1-129 construct, which lacks a PX domain, all mutants retained an endosomal localisation. Scale bars: 20 µm.
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    Mib1 E3 Ligase Interacts with and Destabilizes GABARAP and Promotes GABARAP Ubiquitination at Lys13 and Lys23 (A) HEK293A cells expressing FLAG-Mib1 or control vector for 48 hr were analyzed by immunoblot. (B) Quantification of (A). Statistical analysis using unpaired Student’s t test; mean ± SEM; n = 3; ∗ p ≤ 0.001. (C) Anti-GABARAP immunoprecipitate from HEK293A cells analyzed by immunoblotting. Ab, anti-GABARAP antibody. Lys, HEK293A lysate. (D) HEK293A cells expressing FLAG-tagged constructs were incubated with recombinant GST or GST-GABARAP beads and immunoblotted. (E) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were stringently washed in denaturing buffer. CS, C985S; GAB, GABARAP; Ponc, Ponceau S. Short and long exposures are shown. ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-GABARAP, respectively. (F) Immunoprecipitation of U2OS cells expressing the indicated constructs lysed in boiling SDS buffer and immunoblot. Free ubiquitin and ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-LC3B/GABARAP are indicated, respectively. (G) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were washed as in (E). (H) See (G). Low and high exposures are shown. (I) Immunoprecipitation of HEK293A cells expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 5 mM EDTA) + N-ethylmaleimide. Diubiquitinated GABARAP is indicated with ∗∗ . Immunoglobulin light chain is indicated with an arrow. (J) Immunoprecipitation of HEK293A cells treated with RF or GABARAP siRNA for 72 hr and expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer + N-ethylmaleimide. Di- and tri-ubiquitinated GABARAP is indicated with ∗∗ and ∗∗∗ , respectively. Immunoglobulin light chain is indicated with an arrow. (K) Two GABARAP ubiquitination sites, lysine 13 (K13) and lysine 23 (K23), were identified by mass spectrometry on three different peptides. (Top) Comparison of peak areas for the FVYKEEHPFEK(diGly)R peptide containing K13 ubiquitination site (n = 3 measurements) is shown. (Middle and bottom) The K23 ubiquitination site was detected as two different peptides as a result of missed cleavage. Quantification of peptides K(diGly)KYPDRVPVIVEK (middle) and K(diGly)KYPDR (bottom) showed significantly lower abundance in C985S mutant compared to the WT. (L) Conservation of GABARAP K13 and K23 ( ∗ ) between ATG8 orthologs. See also Figure S5 .

    Journal: Current Biology

    Article Title: Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy

    doi: 10.1016/j.cub.2017.06.021

    Figure Lengend Snippet: Mib1 E3 Ligase Interacts with and Destabilizes GABARAP and Promotes GABARAP Ubiquitination at Lys13 and Lys23 (A) HEK293A cells expressing FLAG-Mib1 or control vector for 48 hr were analyzed by immunoblot. (B) Quantification of (A). Statistical analysis using unpaired Student’s t test; mean ± SEM; n = 3; ∗ p ≤ 0.001. (C) Anti-GABARAP immunoprecipitate from HEK293A cells analyzed by immunoblotting. Ab, anti-GABARAP antibody. Lys, HEK293A lysate. (D) HEK293A cells expressing FLAG-tagged constructs were incubated with recombinant GST or GST-GABARAP beads and immunoblotted. (E) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were stringently washed in denaturing buffer. CS, C985S; GAB, GABARAP; Ponc, Ponceau S. Short and long exposures are shown. ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-GABARAP, respectively. (F) Immunoprecipitation of U2OS cells expressing the indicated constructs lysed in boiling SDS buffer and immunoblot. Free ubiquitin and ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-LC3B/GABARAP are indicated, respectively. (G) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were washed as in (E). (H) See (G). Low and high exposures are shown. (I) Immunoprecipitation of HEK293A cells expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 5 mM EDTA) + N-ethylmaleimide. Diubiquitinated GABARAP is indicated with ∗∗ . Immunoglobulin light chain is indicated with an arrow. (J) Immunoprecipitation of HEK293A cells treated with RF or GABARAP siRNA for 72 hr and expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer + N-ethylmaleimide. Di- and tri-ubiquitinated GABARAP is indicated with ∗∗ and ∗∗∗ , respectively. Immunoglobulin light chain is indicated with an arrow. (K) Two GABARAP ubiquitination sites, lysine 13 (K13) and lysine 23 (K23), were identified by mass spectrometry on three different peptides. (Top) Comparison of peak areas for the FVYKEEHPFEK(diGly)R peptide containing K13 ubiquitination site (n = 3 measurements) is shown. (Middle and bottom) The K23 ubiquitination site was detected as two different peptides as a result of missed cleavage. Quantification of peptides K(diGly)KYPDRVPVIVEK (middle) and K(diGly)KYPDR (bottom) showed significantly lower abundance in C985S mutant compared to the WT. (L) Conservation of GABARAP K13 and K23 ( ∗ ) between ATG8 orthologs. See also Figure S5 .

    Article Snippet: To inhibit deubiquitinases cells were lysed in TNTE buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 1x Complete protease inhibitor (Roche), 1x PhosSTOP (Roche)) supplemented with 20 mM N-Ethylmaleimide (NEM) prior to immunoprecipitation as described.

    Techniques: Expressing, Plasmid Preparation, Construct, Incubation, Recombinant, Immunoprecipitation, Lysis, Mass Spectrometry, Mutagenesis

    Sustained activation of N211Q DDR1b. COS1 cells expressing WT or N211Q DDR1b were serum-starved (18 h) before stimulation (2 h) with (+) 10 μg/ml rat tail collagen I ( Col. I ) or vehicle control (−), as described under “Experimental Procedures.” After stimulation, the media were aspirated, and the cells were washed thoroughly with warm PBS. The dishes were then supplemented with serum-free media and incubated at 37 °C for the indicated times. The cells were lysed with RIPA buffer, and the lysates were analyzed for receptor activation ( A ) and total receptor expression ( B ), as described in Fig. 2 . Black arrow in A indicates phosphorylated DDR1b, and white arrow in B indicates total DDR1b. Anti-Tyr(P) (α- pTyr ).

    Journal: The Journal of Biological Chemistry

    Article Title: Glycosylation at Asn211 Regulates the Activation State of the Discoidin Domain Receptor 1 (DDR1) *

    doi: 10.1074/jbc.M113.541102

    Figure Lengend Snippet: Sustained activation of N211Q DDR1b. COS1 cells expressing WT or N211Q DDR1b were serum-starved (18 h) before stimulation (2 h) with (+) 10 μg/ml rat tail collagen I ( Col. I ) or vehicle control (−), as described under “Experimental Procedures.” After stimulation, the media were aspirated, and the cells were washed thoroughly with warm PBS. The dishes were then supplemented with serum-free media and incubated at 37 °C for the indicated times. The cells were lysed with RIPA buffer, and the lysates were analyzed for receptor activation ( A ) and total receptor expression ( B ), as described in Fig. 2 . Black arrow in A indicates phosphorylated DDR1b, and white arrow in B indicates total DDR1b. Anti-Tyr(P) (α- pTyr ).

    Article Snippet: To obtain the cell lysates, the cells were washed twice with cold PBS and then lysed in RIPA buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with protease inhibitors (Roche Applied Science, complete, Mini, EDTA-free), 10 mm NaF, and 1 mm sodium orthovanadate.

    Techniques: Activation Assay, Expressing, Incubation

    Pea chloroplast FBPase oxidation under atmospheric conditions. A, western blot analysis of the in vitro oxidation of the recombinant FBPase. Recombinant cFBP1 was incubated in a solution with 100 mM Tris-HCl (pH 8.0) and 50 mM DTT. After a 30 min incubation at room temperature free cysteines were blocked by adding four volumes of an alkylating solution containing 100 mM Tris-HCl (pH 8.0), 2.7% CHAPS, and 40 mM IAM (lane 1) or after removing excess DTT by gel filtration before adding the alkylating solution (lane 2). 100 ng of the recombinant protein were loaded per lane. B, western blot analysis of the in vitro oxidation of the chloroplast FBPase present in pea extracts. Leaves soluble proteins (detailed extraction protocol in the Section 2 ) were incubated with DTT and free cysteines alkylated as described in panel A. 10 μg of total soluble protein were loaded per lane. 1, non-reduced protein extract; 2, DTT-incubated protein extract; 3, DTT-incubated protein extract after gel filtration. C, schematic model representing the pea FBPase oxidation exposed to atmospheric air. The green colour represents reduced subunits and the red colour represents oxidized subunits. The structures were prepared using PyMOL Molecular Graphics software ( www.pymol.org ). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Journal: Redox Biology

    Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum

    doi: 10.1016/j.redox.2017.10.008

    Figure Lengend Snippet: Pea chloroplast FBPase oxidation under atmospheric conditions. A, western blot analysis of the in vitro oxidation of the recombinant FBPase. Recombinant cFBP1 was incubated in a solution with 100 mM Tris-HCl (pH 8.0) and 50 mM DTT. After a 30 min incubation at room temperature free cysteines were blocked by adding four volumes of an alkylating solution containing 100 mM Tris-HCl (pH 8.0), 2.7% CHAPS, and 40 mM IAM (lane 1) or after removing excess DTT by gel filtration before adding the alkylating solution (lane 2). 100 ng of the recombinant protein were loaded per lane. B, western blot analysis of the in vitro oxidation of the chloroplast FBPase present in pea extracts. Leaves soluble proteins (detailed extraction protocol in the Section 2 ) were incubated with DTT and free cysteines alkylated as described in panel A. 10 μg of total soluble protein were loaded per lane. 1, non-reduced protein extract; 2, DTT-incubated protein extract; 3, DTT-incubated protein extract after gel filtration. C, schematic model representing the pea FBPase oxidation exposed to atmospheric air. The green colour represents reduced subunits and the red colour represents oxidized subunits. The structures were prepared using PyMOL Molecular Graphics software ( www.pymol.org ). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

    Techniques: Western Blot, In Vitro, Recombinant, Incubation, Filtration, Software

    FBPase oxidation by GSNO incubation. A, western blot analyses of the FBPase oxidation after dark incubation with 0.1 mM of the oxidizing molecules GSNO, oxidized glutathione (GSSG), or hydrogen peroxide (H 2 O 2 ) (in a solution with 100 mM Tris-HCl pH 8.0). No oxidizing compound was added to the control condition. Samples were taken at the indicated times and immediately incubated for 1 h at 37 °C with one volume of alkylating buffer (100 mM Tris-HCl [pH 8.0], 4% CHAPS, and 60 mM IAM). B, effect of GSNO concentration on FBPase oxidation. cFBP1 was incubated as in A, with the indicated GSNO concentrations for 2 h. 100 ng of recombinant FBPase were loaded per lane in A and B. C, inactivation of the FBPase activity following 30 min incubations with GSNO. Activity assays were carried out as described in the Section 2 .

    Journal: Redox Biology

    Article Title: Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum

    doi: 10.1016/j.redox.2017.10.008

    Figure Lengend Snippet: FBPase oxidation by GSNO incubation. A, western blot analyses of the FBPase oxidation after dark incubation with 0.1 mM of the oxidizing molecules GSNO, oxidized glutathione (GSSG), or hydrogen peroxide (H 2 O 2 ) (in a solution with 100 mM Tris-HCl pH 8.0). No oxidizing compound was added to the control condition. Samples were taken at the indicated times and immediately incubated for 1 h at 37 °C with one volume of alkylating buffer (100 mM Tris-HCl [pH 8.0], 4% CHAPS, and 60 mM IAM). B, effect of GSNO concentration on FBPase oxidation. cFBP1 was incubated as in A, with the indicated GSNO concentrations for 2 h. 100 ng of recombinant FBPase were loaded per lane in A and B. C, inactivation of the FBPase activity following 30 min incubations with GSNO. Activity assays were carried out as described in the Section 2 .

    Article Snippet: The assay was done in microtiter plates in a final volume of 200 μl, containing the following components: 2 μg of FBPase, 100 mM Tris-HCl buffer pH (8.0), 1 mM (low Mg2+ ) or 10 mM (high Mg2+ ) MgCl2 , 0.6 mM fructose-1,6-bisphosphate, 0.3 mM NADP+ , 0.7 U phosphoglucose isomerase (ROCHE), and 0.3 U glucose-6-phosphate dehydrogenase (ROCHE).

    Techniques: Incubation, Western Blot, Concentration Assay, Recombinant, Activity Assay

    Co-immunoprecipitation of Htt with SNX21 requires negatively charged residues in the SNX21 N-terminus, but does not require SNX21 to be endosomally localised. (A) Adaptation of SNX20 and SNX21 protein alignment previously generated by Clairfeuille and colleagues ( Clairfeuille et al., 2015 ) . Green boxed regions represent clusters of negatively charged amino acids not present in the SNX20 N-terminal extension. Red boxes denote conserved amino acids within SNX20 and SNX21 sequences and asterisks count every ten residues starting with the fist methionine of SNX20. (B) HEK293-T cells were transiently transfected to express GFP, GFP-tagged full-length SNX21 and two truncation mutants representing the two halves of the N-terminal region of SNX21. Precipitates from the GFP-nanotrap-isolated variants were analysed by western blotting and demonstrate the necessity for a full N-terminal extension in order to facilitate Htt binding. Data are representative of three biological replicates. (C) Site-directed mutagenesis was used to engineer a variety of charge swap mutants targeting the negatively charged clusters of amino acids, prior to probing for Htt binding as above. Two aspartic acid residues in the first N-terminal cluster are essential for precipitation of Htt with SNX21. (D) Both the point mutated GFP-SNX21 and truncation variants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Except for the N-terminal 1-129 construct, which lacks a PX domain, all mutants retained an endosomal localisation. Scale bars: 20 µm.

    Journal: Journal of Cell Science

    Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin

    doi: 10.1242/jcs.211672

    Figure Lengend Snippet: Co-immunoprecipitation of Htt with SNX21 requires negatively charged residues in the SNX21 N-terminus, but does not require SNX21 to be endosomally localised. (A) Adaptation of SNX20 and SNX21 protein alignment previously generated by Clairfeuille and colleagues ( Clairfeuille et al., 2015 ) . Green boxed regions represent clusters of negatively charged amino acids not present in the SNX20 N-terminal extension. Red boxes denote conserved amino acids within SNX20 and SNX21 sequences and asterisks count every ten residues starting with the fist methionine of SNX20. (B) HEK293-T cells were transiently transfected to express GFP, GFP-tagged full-length SNX21 and two truncation mutants representing the two halves of the N-terminal region of SNX21. Precipitates from the GFP-nanotrap-isolated variants were analysed by western blotting and demonstrate the necessity for a full N-terminal extension in order to facilitate Htt binding. Data are representative of three biological replicates. (C) Site-directed mutagenesis was used to engineer a variety of charge swap mutants targeting the negatively charged clusters of amino acids, prior to probing for Htt binding as above. Two aspartic acid residues in the first N-terminal cluster are essential for precipitation of Htt with SNX21. (D) Both the point mutated GFP-SNX21 and truncation variants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Except for the N-terminal 1-129 construct, which lacks a PX domain, all mutants retained an endosomal localisation. Scale bars: 20 µm.

    Article Snippet: Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40).

    Techniques: Immunoprecipitation, Generated, Transfection, Isolation, Western Blot, Binding Assay, Mutagenesis, Construct

    Co-immunoprecipitation of septins with SNX21 requires a surface exposed leucine in the PXB domain. (A) HEK293-T cells were transiently transfected with constructs encoding GFP, GFP-SNX20, GFP-SNX21 and various SNX21 point mutants. After GFP-nanotrap immunoisolation, precipitates were analysed by SDS-PAGE and western blotting. GFP-SNX21 precipitates both septin 2 and septin 7, an interaction that occurs via the SNX21 PXB domain and appears to require the endosomal localisation of SNX21. Data are representative of three biological replicates. (B) Amino acid residues mutated in the current study mapped onto the published structure of the mouse SNX21 ( Clairfeuille et al., 2015 ). (C) Site-directed mutagenesis of the SNX21 PXB domain, targeting predicted surface exposed residues. Constructs encoding GFP-tag chimeras of the various SNX21 mutants were transiently expressed in HEK293-T cells prior to GFP-nanotrap, SDS-PAGE and western blotting. Mutation of an evolutionarily conserved leucine (L363A) and a neighbouring lysine (K364E) is sufficient to perturb association with both septin 2 and septin 7. Data are representative of three biological replicates. (D) GFP-SNX21 mutants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Each of the mutants analysed retained an endosomal localisation in accordance with the wild-type protein. Scale bars: 20 µm.

    Journal: Journal of Cell Science

    Article Title: Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin

    doi: 10.1242/jcs.211672

    Figure Lengend Snippet: Co-immunoprecipitation of septins with SNX21 requires a surface exposed leucine in the PXB domain. (A) HEK293-T cells were transiently transfected with constructs encoding GFP, GFP-SNX20, GFP-SNX21 and various SNX21 point mutants. After GFP-nanotrap immunoisolation, precipitates were analysed by SDS-PAGE and western blotting. GFP-SNX21 precipitates both septin 2 and septin 7, an interaction that occurs via the SNX21 PXB domain and appears to require the endosomal localisation of SNX21. Data are representative of three biological replicates. (B) Amino acid residues mutated in the current study mapped onto the published structure of the mouse SNX21 ( Clairfeuille et al., 2015 ). (C) Site-directed mutagenesis of the SNX21 PXB domain, targeting predicted surface exposed residues. Constructs encoding GFP-tag chimeras of the various SNX21 mutants were transiently expressed in HEK293-T cells prior to GFP-nanotrap, SDS-PAGE and western blotting. Mutation of an evolutionarily conserved leucine (L363A) and a neighbouring lysine (K364E) is sufficient to perturb association with both septin 2 and septin 7. Data are representative of three biological replicates. (D) GFP-SNX21 mutants were expressed in HeLa cells prior to fixation and immunolabelling with anti-EEA1. Each of the mutants analysed retained an endosomal localisation in accordance with the wild-type protein. Scale bars: 20 µm.

    Article Snippet: Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3 VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40).

    Techniques: Immunoprecipitation, Transfection, Construct, SDS Page, Western Blot, Mutagenesis