Structured Review

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Dimerisation of the two human ZnT8 CTD variants. Representative ( n = 3) MST traces for dimerisation of ZnT8 CTD protein. Fluorescently labelled <t>apo‐ZnT8cR</t> (100 n m , magenta circles) was titrated (in the presence of 1 m m <t>EDTA</t> ) with unlabelled apo‐ZnT8cR protein (180 μ m –5.5 n m ), yielding a homodimerisation K d of 4.3 ± 1.3 μ m . Fluorescently labelled apo‐ZnT8cW (100 n m , teal triangles) was titrated (in the presence of 1 m m EDTA ) with unlabelled apo‐ZnT8cW protein (124 μ m –3.8 n m ), with a homodimerisation K d of 1.8 ± 0.1 μ m . There is a significant difference between the homodimerisation K d of each variant in the presence of EDTA ( n = 3, P = 0.034).
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Images

1) Product Images from "The C‐terminal cytosolic domain of the human zinc transporter ZnT8 and its diabetes risk variant"

Article Title: The C‐terminal cytosolic domain of the human zinc transporter ZnT8 and its diabetes risk variant

Journal: The Febs Journal

doi: 10.1111/febs.14402

Dimerisation of the two human ZnT8 CTD variants. Representative ( n = 3) MST traces for dimerisation of ZnT8 CTD protein. Fluorescently labelled apo‐ZnT8cR (100 n m , magenta circles) was titrated (in the presence of 1 m m EDTA ) with unlabelled apo‐ZnT8cR protein (180 μ m –5.5 n m ), yielding a homodimerisation K d of 4.3 ± 1.3 μ m . Fluorescently labelled apo‐ZnT8cW (100 n m , teal triangles) was titrated (in the presence of 1 m m EDTA ) with unlabelled apo‐ZnT8cW protein (124 μ m –3.8 n m ), with a homodimerisation K d of 1.8 ± 0.1 μ m . There is a significant difference between the homodimerisation K d of each variant in the presence of EDTA ( n = 3, P = 0.034).
Figure Legend Snippet: Dimerisation of the two human ZnT8 CTD variants. Representative ( n = 3) MST traces for dimerisation of ZnT8 CTD protein. Fluorescently labelled apo‐ZnT8cR (100 n m , magenta circles) was titrated (in the presence of 1 m m EDTA ) with unlabelled apo‐ZnT8cR protein (180 μ m –5.5 n m ), yielding a homodimerisation K d of 4.3 ± 1.3 μ m . Fluorescently labelled apo‐ZnT8cW (100 n m , teal triangles) was titrated (in the presence of 1 m m EDTA ) with unlabelled apo‐ZnT8cW protein (124 μ m –3.8 n m ), with a homodimerisation K d of 1.8 ± 0.1 μ m . There is a significant difference between the homodimerisation K d of each variant in the presence of EDTA ( n = 3, P = 0.034).

Techniques Used: Microscale Thermophoresis, Variant Assay

2) Product Images from "Altered protease and antiprotease balance during a COPD exacerbation contributes to mucus obstruction"

Article Title: Altered protease and antiprotease balance during a COPD exacerbation contributes to mucus obstruction

Journal: Respiratory Research

doi: 10.1186/s12931-015-0247-x

Serine proteases inhibit mucin degradation. Analysis of sputum MUC5AC and MUC5B by western blot after incubation at 37 °C over 24 h with or without of protease inhibitors. Sputum was obtained from a COPD subject 5–6 weeks after an acute exacerbation. Mucin concentration of the native control without incubation over 24 h was set to 100 %. a We used the serine protease inhibitors DFP, PMSF and TLCK, the metalloprotease inhibitors EDTA and GM6001 and the cysteine protease inhibitors leupeptin and E64. Analysis was performed in triplicate. b Incubation of COPD sputa (5–6 weeks after the onset) with A1-PI ( n = 4) and compared with control sputa
Figure Legend Snippet: Serine proteases inhibit mucin degradation. Analysis of sputum MUC5AC and MUC5B by western blot after incubation at 37 °C over 24 h with or without of protease inhibitors. Sputum was obtained from a COPD subject 5–6 weeks after an acute exacerbation. Mucin concentration of the native control without incubation over 24 h was set to 100 %. a We used the serine protease inhibitors DFP, PMSF and TLCK, the metalloprotease inhibitors EDTA and GM6001 and the cysteine protease inhibitors leupeptin and E64. Analysis was performed in triplicate. b Incubation of COPD sputa (5–6 weeks after the onset) with A1-PI ( n = 4) and compared with control sputa

Techniques Used: Western Blot, Incubation, Concentration Assay

3) Product Images from "Serine Proteases Degrade Airway Mucins in Cystic Fibrosis ▿Serine Proteases Degrade Airway Mucins in Cystic Fibrosis ▿ †"

Article Title: Serine Proteases Degrade Airway Mucins in Cystic Fibrosis ▿Serine Proteases Degrade Airway Mucins in Cystic Fibrosis ▿ †

Journal: Infection and Immunity

doi: 10.1128/IAI.01252-10

Analysis of sputum MUC5AC and MUC5B by Western blotting after incubation at 37°C over 6 h in the presence or absence of proteases inhibitors. Sputum was obtained from a CF subject chronically infected by P. aeruginosa . The mucin concentration of the native control without incubation was set to 100%, and results are presented relative to this. We used the serine protease inhibitors DFP, PMSF, and TLCK, the metalloprotease inhibitors EDTA and GM6001, and the cysteine protease inhibitors leupeptin and E64.
Figure Legend Snippet: Analysis of sputum MUC5AC and MUC5B by Western blotting after incubation at 37°C over 6 h in the presence or absence of proteases inhibitors. Sputum was obtained from a CF subject chronically infected by P. aeruginosa . The mucin concentration of the native control without incubation was set to 100%, and results are presented relative to this. We used the serine protease inhibitors DFP, PMSF, and TLCK, the metalloprotease inhibitors EDTA and GM6001, and the cysteine protease inhibitors leupeptin and E64.

Techniques Used: Western Blot, Incubation, Infection, Concentration Assay

4) Product Images from "The amyloid fold of Gad m 1 epitopes governs IgE binding"

Article Title: The amyloid fold of Gad m 1 epitopes governs IgE binding

Journal: Scientific Reports

doi: 10.1038/srep32801

Gad m 1 sequences that are recognized by IgE in patient sera display amyloid folds. (a) The effects of patient sera (S-I/II) and the anti-amyloid OC antibody on the amyloid formation kinetics of rGad wt (black), its ACE mutant (orange) and Aβ42 (green) were monitored by ThT-binding in 50 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl and 5 mM EDTA. (b) Effects of the anti-amyloid OC antibody and Gad m 1 monomers on recognition of the wt and ACE amyloid fibrils by the IgE in patient sera, as determined by the ELISA assay. Amyloid fibrils (100 ng/well) were probed with the patient sera pool that had been pre-adsorbed with 1 μg of BSA (S-I/II), 1 μg of OC (OC-preadsorbed S-I/II) and 1 μg of the corresponding monomer chain (M-preadsorbed S-I/II). (c) Gad m1 peptide arrays were probed with amyloids (wt-F and ACE-F) and monomers (ACE-M) and developed with the anti-His tag antibody to detect binding. (d) Sequence coverage of rGad m 1 wt obtained by LC-MS/MS analysis of the peptide mixture after pepsin digestion of the amyloids. The pepsin cleavage sites are displayed in red, the predicted adhesive regions are shown as blue lines, and the IgE binding regions are indicated with green lines (solid in the common core, dotted for all peptides). The sequences of the overlapping IgE-binding regions in the peptides are depicted in pink and their mass/charge ratio (m/z) is indicated nearby.
Figure Legend Snippet: Gad m 1 sequences that are recognized by IgE in patient sera display amyloid folds. (a) The effects of patient sera (S-I/II) and the anti-amyloid OC antibody on the amyloid formation kinetics of rGad wt (black), its ACE mutant (orange) and Aβ42 (green) were monitored by ThT-binding in 50 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl and 5 mM EDTA. (b) Effects of the anti-amyloid OC antibody and Gad m 1 monomers on recognition of the wt and ACE amyloid fibrils by the IgE in patient sera, as determined by the ELISA assay. Amyloid fibrils (100 ng/well) were probed with the patient sera pool that had been pre-adsorbed with 1 μg of BSA (S-I/II), 1 μg of OC (OC-preadsorbed S-I/II) and 1 μg of the corresponding monomer chain (M-preadsorbed S-I/II). (c) Gad m1 peptide arrays were probed with amyloids (wt-F and ACE-F) and monomers (ACE-M) and developed with the anti-His tag antibody to detect binding. (d) Sequence coverage of rGad m 1 wt obtained by LC-MS/MS analysis of the peptide mixture after pepsin digestion of the amyloids. The pepsin cleavage sites are displayed in red, the predicted adhesive regions are shown as blue lines, and the IgE binding regions are indicated with green lines (solid in the common core, dotted for all peptides). The sequences of the overlapping IgE-binding regions in the peptides are depicted in pink and their mass/charge ratio (m/z) is indicated nearby.

Techniques Used: Mutagenesis, Binding Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Related Articles

Clone Assay:

Article Title: The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6
Article Snippet: After expression overnight at 22°C upon induction with 1 mM IPTG, the cultures were chemically lysed (0.4 M boric acid buffer, pH 8.0 containing 320 mM NaCl, 4 mM EDTA, 0.25% (w/v) lysozyme and 12.5 U ml−1 Benzonase® (Merck Millipore)) and the crude extracts were tested via ELISA for the presence of specifically binding antibody fragments. .. For all clones exhibiting a strong binding signal for the antigens (greater than or equal to fivefold over background) the DNA sequence encoding the complementarity-determining regions (CDRs) of the antibody variable heavy chain was determined.

Centrifugation:

Article Title: Protein Profiling Gastric Cancer and Neighboring Control Tissues Using High-Content Antibody Microarrays
Article Snippet: The material was lysed for 30 min on ice in 10 volumes of lysis buffer composed of 50 mM carbonate buffer (pH 8.5), 20% glycerol, 1.0 mM MgCl2 , 5.0 mM EDTA, 1.0 mM phenylmethanesulfonyl fluoride, 1.0 U/mL benzonase (Merck Biosciences, Schwalbach, Germany), Halt protease and phosphatase inhibitor mixture (Thermo Scientific, Bonn, Germany), 0.5% Nonidet P-40 substitute, 1.0% cholic acid, 0.25% n-dodecyl-maltoside (GenaXXon Bioscience, Ulm, Germany), and 0.5% amidosulfobetaine-14. .. Cell debris was pelleted by 20 min centrifugation at 4 °C and 13,000 rpm.

Article Title: Cell-sized confinement controls generation and stability of a protein wave for spatiotemporal regulation in cells
Article Snippet: The collected cells were disrupted by sonication using a Sonifier250 (Branson, Danbury, CT, USA), and the supernatant of the crude extract was fractionated by centrifugation at 20,000 g at 4°C for 30 min. To purify His-tagged proteins, the crude extracts mixed with cOmplete His-Tag purification resin (Roche, Basel, Switzerland) were loaded onto a polyprep chromatography column (Bio-Rad, Hercules, CA, USA), and washed with 25 mL WS buffer [50 mM NaH2 PO4 (pH 7.6), 300 mM NaCl, 20 mM imidazole, 10% glycerol, 0.1 mM EDTA, and 0.1 mM PMSF]. .. EL buffer was exchanged with storage buffer [50 mM HEPES-KOH (pH 7.6), 150 mM GluK, 10% glycerol, 0.1 mM EDTA] with 0.2 mM ADP-Mg by ultrafiltration using AmiconUltra-15 10 k and AmiconUltra-0.5 30 k filters (Merck Millipore).

Article Title: The C‐terminal cytosolic domain of the human zinc transporter ZnT8 and its diabetes risk variant
Article Snippet: The 60 mL supernatant was added to 2 mL of pre‐equilibrated Ni2+ affinity gel (Sigma Aldrich) and incubated end‐over‐end on a roller at 4 °C for 40 min. After centrifugation at 500 g for 1 min at 4 °C, the pellet was washed three times with equilibration buffer containing 500 mm NaCl. .. To produce ZnT8c apo‐protein, 2 mm TCEP and 1 mm EDTA were added to the 10 mL fractions from size exclusion chromatography and then concentrated to ~ 0.5 mL using a 15 mL 3 kDa MWCO centrifugal concentrator (Merck Millipore).

Article Title: Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts
Article Snippet: .. Cells were collected by centrifugation (6000 × g , 10 min) and resuspended in 25 mL TEMB buffer (5 mM Tris-HCl [pH 8.0], 1 mM EDTA, 10 mM MgCl2 , 20 mM NaHCO3 ) containing 0.55 M mannitol and 60 kU rLysozyme (Merck-Millipore, Bayswater, VIC, Australia). .. Cells were incubated at 37 °C in the dark for 2–16 h with gentle shaking to enable cell wall degradation and then collected by centrifugation as above.

Synthesized:

Article Title: The amyloid fold of Gad m 1 epitopes governs IgE binding
Article Snippet: Peptide arrays (SPOT) Dodecapeptides spanning the whole sequence of Gad m 1, with an overlap of ten residues, were solid-phase synthesized and immobilized (≈10 nmol per spot) on an Amino-PEG500-UC540 sheet at the National Centre for Biotechnology (CNB-CSIC, Madrid). .. Before use, the membranes were rinsed with ethanol, washed three times with TBS (25 mM Tris-HCl, pH 7.5, containing 137 mM NaCl and 2.7 mM KCl), and incubated in TBS containing 1% BSA (w/v) and 2 mM EDTA for 1 h. The membranes were then probed for 2 h with sera from patients who are allergic to fish (1/10 dilution) and the anti-amyloid fibril OC antibody (AB2286 Merck Millipore, 1/2,000 dilution), prepared in TBST (TBS containing 0.05% Tween-20) supplemented with 0.5% BSA (w/v) or with rGad m 1 chains (2.5 μM) in TBS.

Enzyme-linked Immunosorbent Assay:

Article Title: The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6
Article Snippet: .. After expression overnight at 22°C upon induction with 1 mM IPTG, the cultures were chemically lysed (0.4 M boric acid buffer, pH 8.0 containing 320 mM NaCl, 4 mM EDTA, 0.25% (w/v) lysozyme and 12.5 U ml−1 Benzonase® (Merck Millipore)) and the crude extracts were tested via ELISA for the presence of specifically binding antibody fragments. .. For all clones exhibiting a strong binding signal for the antigens (greater than or equal to fivefold over background) the DNA sequence encoding the complementarity-determining regions (CDRs) of the antibody variable heavy chain was determined.

Incubation:

Article Title: Serine Proteases Degrade Airway Mucins in Cystic Fibrosis ▿Serine Proteases Degrade Airway Mucins in Cystic Fibrosis ▿ †
Article Snippet: To assess whether mucin degradation in the CF sputum could be inhibited by a protease inhibitor, we incubated the sputum at 37°C over 6 h in the absence or presence of protease inhibitors. .. Chemicals were purchased from Sigma (St. Louis, MO) (DFP [final concentration, 2 mM], PMSF [final concentration, 2 mM], TLCK [final concentration, 10 mM], EDTA [final concentration, 100 mM], and E64 [final concentration, 500 ng/ml]) or Merck Chemical (Nottingham, United Kingdom) (GM6001 [final concentration, 40 μM] and leupeptin [final concentration, 40 μM]).

Article Title: The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6
Article Snippet: In each panning round, the antibody phage library was incubated with biotinylated sclerostin protein, binding Fabs were captured via streptavidin-coated magnetic beads (Invitrogen) and phages were eluted with 25 mM DTT. .. After expression overnight at 22°C upon induction with 1 mM IPTG, the cultures were chemically lysed (0.4 M boric acid buffer, pH 8.0 containing 320 mM NaCl, 4 mM EDTA, 0.25% (w/v) lysozyme and 12.5 U ml−1 Benzonase® (Merck Millipore)) and the crude extracts were tested via ELISA for the presence of specifically binding antibody fragments.

Article Title: The C‐terminal cytosolic domain of the human zinc transporter ZnT8 and its diabetes risk variant
Article Snippet: The 60 mL supernatant was added to 2 mL of pre‐equilibrated Ni2+ affinity gel (Sigma Aldrich) and incubated end‐over‐end on a roller at 4 °C for 40 min. After centrifugation at 500 g for 1 min at 4 °C, the pellet was washed three times with equilibration buffer containing 500 mm NaCl. .. To produce ZnT8c apo‐protein, 2 mm TCEP and 1 mm EDTA were added to the 10 mL fractions from size exclusion chromatography and then concentrated to ~ 0.5 mL using a 15 mL 3 kDa MWCO centrifugal concentrator (Merck Millipore).

Article Title: The amyloid fold of Gad m 1 epitopes governs IgE binding
Article Snippet: .. Before use, the membranes were rinsed with ethanol, washed three times with TBS (25 mM Tris-HCl, pH 7.5, containing 137 mM NaCl and 2.7 mM KCl), and incubated in TBS containing 1% BSA (w/v) and 2 mM EDTA for 1 h. The membranes were then probed for 2 h with sera from patients who are allergic to fish (1/10 dilution) and the anti-amyloid fibril OC antibody (AB2286 Merck Millipore, 1/2,000 dilution), prepared in TBST (TBS containing 0.05% Tween-20) supplemented with 0.5% BSA (w/v) or with rGad m 1 chains (2.5 μM) in TBS. .. After extensive washes with TBST, a 30 min incubation with either horseradish peroxidase-labeled anti-human IgE (Abcam ab99806, 1/2,000 dilution), anti-human IgG4 (Abcam ab99823, 1/4,000 dilution), goat anti-rabbit IgG (Sigma, 1/5,000 dilution) or anti-6X His tag® antibody (Abcam ab18184, dilution 1/1,000) was performed.

Article Title: Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts
Article Snippet: Cells were collected by centrifugation (6000 × g , 10 min) and resuspended in 25 mL TEMB buffer (5 mM Tris-HCl [pH 8.0], 1 mM EDTA, 10 mM MgCl2 , 20 mM NaHCO3 ) containing 0.55 M mannitol and 60 kU rLysozyme (Merck-Millipore, Bayswater, VIC, Australia). .. Cells were incubated at 37 °C in the dark for 2–16 h with gentle shaking to enable cell wall degradation and then collected by centrifugation as above.

Expressing:

Article Title: Cell-sized confinement controls generation and stability of a protein wave for spatiotemporal regulation in cells
Article Snippet: Paragraph title: Expression and purification of MinD and its mutant ... EL buffer was exchanged with storage buffer [50 mM HEPES-KOH (pH 7.6), 150 mM GluK, 10% glycerol, 0.1 mM EDTA] with 0.2 mM ADP-Mg by ultrafiltration using AmiconUltra-15 10 k and AmiconUltra-0.5 30 k filters (Merck Millipore).

Article Title: The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6
Article Snippet: .. After expression overnight at 22°C upon induction with 1 mM IPTG, the cultures were chemically lysed (0.4 M boric acid buffer, pH 8.0 containing 320 mM NaCl, 4 mM EDTA, 0.25% (w/v) lysozyme and 12.5 U ml−1 Benzonase® (Merck Millipore)) and the crude extracts were tested via ELISA for the presence of specifically binding antibody fragments. .. For all clones exhibiting a strong binding signal for the antigens (greater than or equal to fivefold over background) the DNA sequence encoding the complementarity-determining regions (CDRs) of the antibody variable heavy chain was determined.

Article Title: The C‐terminal cytosolic domain of the human zinc transporter ZnT8 and its diabetes risk variant
Article Snippet: Paragraph title: Protein expression and purification ... To produce ZnT8c apo‐protein, 2 mm TCEP and 1 mm EDTA were added to the 10 mL fractions from size exclusion chromatography and then concentrated to ~ 0.5 mL using a 15 mL 3 kDa MWCO centrifugal concentrator (Merck Millipore).

BIA-KA:

Article Title: Protein Profiling Gastric Cancer and Neighboring Control Tissues Using High-Content Antibody Microarrays
Article Snippet: The material was lysed for 30 min on ice in 10 volumes of lysis buffer composed of 50 mM carbonate buffer (pH 8.5), 20% glycerol, 1.0 mM MgCl2 , 5.0 mM EDTA, 1.0 mM phenylmethanesulfonyl fluoride, 1.0 U/mL benzonase (Merck Biosciences, Schwalbach, Germany), Halt protease and phosphatase inhibitor mixture (Thermo Scientific, Bonn, Germany), 0.5% Nonidet P-40 substitute, 1.0% cholic acid, 0.25% n-dodecyl-maltoside (GenaXXon Bioscience, Ulm, Germany), and 0.5% amidosulfobetaine-14. .. The protein concentration was determined with the Bicinchoninic Acid Protein Assay Reagent (BCA) kit (Thermo Scientific) according to the manufacturer’s protocol.

Modification:

Article Title: Evaluation of Potential Effects of NaCl and Sorbic Acid on Staphylococcal Enterotoxin A Formation
Article Snippet: RNA Extraction A modified version of the protocol described by Lövenklev et al . .. For RNA extraction, each sample pellet was resuspended in 500 µL ice cold TES buffer, pH 7.5 (50 mM Tris (BDH Prolabo, VWR International, Stockholm, Sweden), 5 mM EDTA and 50 mM NaCl (Merck Millipore)) and the suspension was transferred to Precelly lysing kit tubes (VK 0.1) (Bertin Technologies, Montigny-le-Bretonneux, France).

Transformation Assay:

Article Title: Cell-sized confinement controls generation and stability of a protein wave for spatiotemporal regulation in cells
Article Snippet: E. coli BL21-CodonPlus(DE3)-RIPL (Agilent Technologies, Santa Clara, CA, USA) cells were transformed with the resultant plasmids. .. EL buffer was exchanged with storage buffer [50 mM HEPES-KOH (pH 7.6), 150 mM GluK, 10% glycerol, 0.1 mM EDTA] with 0.2 mM ADP-Mg by ultrafiltration using AmiconUltra-15 10 k and AmiconUltra-0.5 30 k filters (Merck Millipore).

Article Title: The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6
Article Snippet: For screening of the antibodies, E. coli of the strain TG1F− (TG1 without the F-plasmid) were transformed and individual colonies were randomly picked and grown in micro-titre plates. .. After expression overnight at 22°C upon induction with 1 mM IPTG, the cultures were chemically lysed (0.4 M boric acid buffer, pH 8.0 containing 320 mM NaCl, 4 mM EDTA, 0.25% (w/v) lysozyme and 12.5 U ml−1 Benzonase® (Merck Millipore)) and the crude extracts were tested via ELISA for the presence of specifically binding antibody fragments.

Flow Cytometry:

Article Title: The C‐terminal cytosolic domain of the human zinc transporter ZnT8 and its diabetes risk variant
Article Snippet: The eluate was loaded onto a Superdex S75 26/60 column (GE Healthcare, Chicago, IL, USA) equilibrated with purification buffer (50 mm Tris/HCl, pH 8, 300 mm NaCl, 100 mm sucrose, 100 μm TCEP) using a flow of 2.2 mL·min−1 and a column temperature of 4 °C. .. To produce ZnT8c apo‐protein, 2 mm TCEP and 1 mm EDTA were added to the 10 mL fractions from size exclusion chromatography and then concentrated to ~ 0.5 mL using a 15 mL 3 kDa MWCO centrifugal concentrator (Merck Millipore).

Chromatography:

Article Title: Cell-sized confinement controls generation and stability of a protein wave for spatiotemporal regulation in cells
Article Snippet: The collected cells were disrupted by sonication using a Sonifier250 (Branson, Danbury, CT, USA), and the supernatant of the crude extract was fractionated by centrifugation at 20,000 g at 4°C for 30 min. To purify His-tagged proteins, the crude extracts mixed with cOmplete His-Tag purification resin (Roche, Basel, Switzerland) were loaded onto a polyprep chromatography column (Bio-Rad, Hercules, CA, USA), and washed with 25 mL WS buffer [50 mM NaH2 PO4 (pH 7.6), 300 mM NaCl, 20 mM imidazole, 10% glycerol, 0.1 mM EDTA, and 0.1 mM PMSF]. .. EL buffer was exchanged with storage buffer [50 mM HEPES-KOH (pH 7.6), 150 mM GluK, 10% glycerol, 0.1 mM EDTA] with 0.2 mM ADP-Mg by ultrafiltration using AmiconUltra-15 10 k and AmiconUltra-0.5 30 k filters (Merck Millipore).

Protease Inhibitor:

Article Title: Serine Proteases Degrade Airway Mucins in Cystic Fibrosis ▿Serine Proteases Degrade Airway Mucins in Cystic Fibrosis ▿ †
Article Snippet: To assess whether mucin degradation in the CF sputum could be inhibited by a protease inhibitor, we incubated the sputum at 37°C over 6 h in the absence or presence of protease inhibitors. .. Chemicals were purchased from Sigma (St. Louis, MO) (DFP [final concentration, 2 mM], PMSF [final concentration, 2 mM], TLCK [final concentration, 10 mM], EDTA [final concentration, 100 mM], and E64 [final concentration, 500 ng/ml]) or Merck Chemical (Nottingham, United Kingdom) (GM6001 [final concentration, 40 μM] and leupeptin [final concentration, 40 μM]).

Article Title: Altered protease and antiprotease balance during a COPD exacerbation contributes to mucus obstruction
Article Snippet: Alpha-1 protease inhibitor (A1-PI) was obtained as Prolastin® (Grifols Therapeutics Inc. Frankfurt, Germany) and was used at a final concentration of 0.3 μg/mL. .. DFP (final concentration 2 mM); PMSF (final concentration 2 mM); TLCK (final concentration 10 mM); EDTA (final concentration 100 mM); E64 (final concentration 500 ng/mL) or Merck Chemical (Nottingham, UK): GM6001 (final concentration 40 μM) and leupeptin (final concentration 40 μM) were used.

Light Microscopy:

Article Title: Babesiosis due to the canine Babesia microti-like small piroplasm in dogs - first report from Portugal and possible vertical transmission
Article Snippet: .. Blood in EDTA was used to prepare thin glass-slide smears that were air-dried, fixed with methanol, stained with Hemacolor® (Merck, Germany) and then examined under light microscopy (magnification of 1000×) for the detection of possible piroplasms. .. Blood was also spotted onto individual papers (7.5 cm × 2.5 cm; GB 002 Schleicher and Schuell, Dassel, Germany) allowed to air-dry and stored at -20°C until further use.

Generated:

Article Title: Altered protease and antiprotease balance during a COPD exacerbation contributes to mucus obstruction
Article Snippet: DFP (final concentration 2 mM); PMSF (final concentration 2 mM); TLCK (final concentration 10 mM); EDTA (final concentration 100 mM); E64 (final concentration 500 ng/mL) or Merck Chemical (Nottingham, UK): GM6001 (final concentration 40 μM) and leupeptin (final concentration 40 μM) were used. .. Polyclonal anti-MUC5AC and anti-MUC5B antibodies were generated as previously described [ ].

other:

Article Title: In Vitro Analysis of CsA-Induced Hepatotoxicity in HepG2 Cell Line: Oxidative Stress and ?2 and ?1 Integrin Subunits Expression
Article Snippet: Sodium azide, sodium chloride, magnesium chloride and EDTA were obtained from Merck (Darmstadt, Germany).

Article Title: Identification of tumour-related proteins as potential screening markers by proteome analysis—protein profiles of human saliva as a predictive and prognostic tool
Article Snippet: CHAPS was obtained from Roche Diagnostics (Mannheim, Germany), urea from AppliChem (Darmstadt, Germany), thiourea from Fluka (Buchs, Switzerland), 1,4-dithioerythritol (DTE) and EDTA from Merck (Darmstadt, Germany) and tributylphosphine (TBP) from Pierce Biotechnology (Rockford, IL, USA).

Article Title: Validation of ethnopharmacological uses of Heliotropium strigosum Willd. as spasmolytic, bronchodilator and vasorelaxant remedy
Article Snippet: Methanol, EDTA, glucose, magnesium chloride, magnesium sulfate, phenylephrine, potassium dihydrogen phosphate, sodium bicarbonate, sodium dihydrogen phosphate and calcium chloride were purchased from Merck, Darmstadt, Germany.

Article Title: Optimization of DNA Extraction for RAPD and ISSR Analysis of Arbutus unedo L. Leaves
Article Snippet: Reagents and Solutions The extraction buffer consisted of 3% (w/v) CTAB (Sigma, Sintra, Portugal), 100 mM Tris-HCl pH 8.0 (CalBiochem, Lisbon, Portugal), 20 mM EDTA pH 8.0 (Merck, Lisbon, Portugal) and 2 M sodium chloride (NaCl; Merck, Lisbon, Portugal).

Protein Concentration:

Article Title: Protein Profiling Gastric Cancer and Neighboring Control Tissues Using High-Content Antibody Microarrays
Article Snippet: The material was lysed for 30 min on ice in 10 volumes of lysis buffer composed of 50 mM carbonate buffer (pH 8.5), 20% glycerol, 1.0 mM MgCl2 , 5.0 mM EDTA, 1.0 mM phenylmethanesulfonyl fluoride, 1.0 U/mL benzonase (Merck Biosciences, Schwalbach, Germany), Halt protease and phosphatase inhibitor mixture (Thermo Scientific, Bonn, Germany), 0.5% Nonidet P-40 substitute, 1.0% cholic acid, 0.25% n-dodecyl-maltoside (GenaXXon Bioscience, Ulm, Germany), and 0.5% amidosulfobetaine-14. .. The protein concentration was determined with the Bicinchoninic Acid Protein Assay Reagent (BCA) kit (Thermo Scientific) according to the manufacturer’s protocol.

Sequencing:

Article Title: The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6
Article Snippet: After expression overnight at 22°C upon induction with 1 mM IPTG, the cultures were chemically lysed (0.4 M boric acid buffer, pH 8.0 containing 320 mM NaCl, 4 mM EDTA, 0.25% (w/v) lysozyme and 12.5 U ml−1 Benzonase® (Merck Millipore)) and the crude extracts were tested via ELISA for the presence of specifically binding antibody fragments. .. For all clones exhibiting a strong binding signal for the antigens (greater than or equal to fivefold over background) the DNA sequence encoding the complementarity-determining regions (CDRs) of the antibody variable heavy chain was determined.

Article Title: The amyloid fold of Gad m 1 epitopes governs IgE binding
Article Snippet: Peptide arrays (SPOT) Dodecapeptides spanning the whole sequence of Gad m 1, with an overlap of ten residues, were solid-phase synthesized and immobilized (≈10 nmol per spot) on an Amino-PEG500-UC540 sheet at the National Centre for Biotechnology (CNB-CSIC, Madrid). .. Before use, the membranes were rinsed with ethanol, washed three times with TBS (25 mM Tris-HCl, pH 7.5, containing 137 mM NaCl and 2.7 mM KCl), and incubated in TBS containing 1% BSA (w/v) and 2 mM EDTA for 1 h. The membranes were then probed for 2 h with sera from patients who are allergic to fish (1/10 dilution) and the anti-amyloid fibril OC antibody (AB2286 Merck Millipore, 1/2,000 dilution), prepared in TBST (TBS containing 0.05% Tween-20) supplemented with 0.5% BSA (w/v) or with rGad m 1 chains (2.5 μM) in TBS.

Sonication:

Article Title: Cell-sized confinement controls generation and stability of a protein wave for spatiotemporal regulation in cells
Article Snippet: The collected cells were disrupted by sonication using a Sonifier250 (Branson, Danbury, CT, USA), and the supernatant of the crude extract was fractionated by centrifugation at 20,000 g at 4°C for 30 min. To purify His-tagged proteins, the crude extracts mixed with cOmplete His-Tag purification resin (Roche, Basel, Switzerland) were loaded onto a polyprep chromatography column (Bio-Rad, Hercules, CA, USA), and washed with 25 mL WS buffer [50 mM NaH2 PO4 (pH 7.6), 300 mM NaCl, 20 mM imidazole, 10% glycerol, 0.1 mM EDTA, and 0.1 mM PMSF]. .. EL buffer was exchanged with storage buffer [50 mM HEPES-KOH (pH 7.6), 150 mM GluK, 10% glycerol, 0.1 mM EDTA] with 0.2 mM ADP-Mg by ultrafiltration using AmiconUltra-15 10 k and AmiconUltra-0.5 30 k filters (Merck Millipore).

Article Title: The C‐terminal cytosolic domain of the human zinc transporter ZnT8 and its diabetes risk variant
Article Snippet: The homogenate was diluted 1 : 6 with equilibration buffer [50 mm Tris/HCl, pH 8, 100 mm NaCl, 100 mm sucrose, 2 mm DTT, 20 mm imidazole, containing one tablet of Complete ULTRA mini EDTA‐free protease inhibitors (Roche, Basel, Switzerland)], and sonicated (Model 2000U, Ultrasonic Power Corp. (Freeport, IL, USA); +285 output, 0.5 s−1 pulse) in an ice‐water bath for 20 s pulse and 40 s rest settings for a total of 15 min, followed by centrifugation at 45 000 g for 40 min at 4 °C. .. To produce ZnT8c apo‐protein, 2 mm TCEP and 1 mm EDTA were added to the 10 mL fractions from size exclusion chromatography and then concentrated to ~ 0.5 mL using a 15 mL 3 kDa MWCO centrifugal concentrator (Merck Millipore).

Injection:

Article Title: Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase
Article Snippet: All ITC measurements reported here were made in this buffer, using the following chemicals: glycine, ( > 99.7%, Merck, Darmstadt, Germany), ethylenediaminetetraacetic acid, EDTA ( > 99%, Merck, Darmstadt, Germany), and SDS > 99%, Fluka, Buchs, Switzerland) and a MCS-ITC (MicroCal, Northampton, MA) isothermal titration calorimeter. .. The cell solution was titrated with 55 aliquots of 5 µL (first injection only 1 µL) of 1.5 m M SDS in the same buffer solution at 38°C.

Binding Assay:

Article Title: The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6
Article Snippet: .. After expression overnight at 22°C upon induction with 1 mM IPTG, the cultures were chemically lysed (0.4 M boric acid buffer, pH 8.0 containing 320 mM NaCl, 4 mM EDTA, 0.25% (w/v) lysozyme and 12.5 U ml−1 Benzonase® (Merck Millipore)) and the crude extracts were tested via ELISA for the presence of specifically binding antibody fragments. .. For all clones exhibiting a strong binding signal for the antigens (greater than or equal to fivefold over background) the DNA sequence encoding the complementarity-determining regions (CDRs) of the antibody variable heavy chain was determined.

Pull Down Assay:

Article Title: Cell-sized confinement controls generation and stability of a protein wave for spatiotemporal regulation in cells
Article Snippet: EL buffer was exchanged with storage buffer [50 mM HEPES-KOH (pH 7.6), 150 mM GluK, 10% glycerol, 0.1 mM EDTA] with 0.2 mM ADP-Mg by ultrafiltration using AmiconUltra-15 10 k and AmiconUltra-0.5 30 k filters (Merck Millipore). .. For pull-down assay, His-sfGFP-MinDD40A Δ10 was treated with thrombin (Wako, Osaka Japan) in the storage buffer at 4°C overnight.

Magnetic Beads:

Article Title: The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6
Article Snippet: In each panning round, the antibody phage library was incubated with biotinylated sclerostin protein, binding Fabs were captured via streptavidin-coated magnetic beads (Invitrogen) and phages were eluted with 25 mM DTT. .. After expression overnight at 22°C upon induction with 1 mM IPTG, the cultures were chemically lysed (0.4 M boric acid buffer, pH 8.0 containing 320 mM NaCl, 4 mM EDTA, 0.25% (w/v) lysozyme and 12.5 U ml−1 Benzonase® (Merck Millipore)) and the crude extracts were tested via ELISA for the presence of specifically binding antibody fragments.

Mutagenesis:

Article Title: Cell-sized confinement controls generation and stability of a protein wave for spatiotemporal regulation in cells
Article Snippet: Paragraph title: Expression and purification of MinD and its mutant ... EL buffer was exchanged with storage buffer [50 mM HEPES-KOH (pH 7.6), 150 mM GluK, 10% glycerol, 0.1 mM EDTA] with 0.2 mM ADP-Mg by ultrafiltration using AmiconUltra-15 10 k and AmiconUltra-0.5 30 k filters (Merck Millipore).

Isolation:

Article Title: Protein Profiling Gastric Cancer and Neighboring Control Tissues Using High-Content Antibody Microarrays
Article Snippet: Paragraph title: 2.1. Protein Isolation and Labeling ... The material was lysed for 30 min on ice in 10 volumes of lysis buffer composed of 50 mM carbonate buffer (pH 8.5), 20% glycerol, 1.0 mM MgCl2 , 5.0 mM EDTA, 1.0 mM phenylmethanesulfonyl fluoride, 1.0 U/mL benzonase (Merck Biosciences, Schwalbach, Germany), Halt protease and phosphatase inhibitor mixture (Thermo Scientific, Bonn, Germany), 0.5% Nonidet P-40 substitute, 1.0% cholic acid, 0.25% n-dodecyl-maltoside (GenaXXon Bioscience, Ulm, Germany), and 0.5% amidosulfobetaine-14.

Article Title: Evaluation of Potential Effects of NaCl and Sorbic Acid on Staphylococcal Enterotoxin A Formation
Article Snippet: For RNA extraction, each sample pellet was resuspended in 500 µL ice cold TES buffer, pH 7.5 (50 mM Tris (BDH Prolabo, VWR International, Stockholm, Sweden), 5 mM EDTA and 50 mM NaCl (Merck Millipore)) and the suspension was transferred to Precelly lysing kit tubes (VK 0.1) (Bertin Technologies, Montigny-le-Bretonneux, France). .. RNA was isolated using two steps of phenol-chloroform (600 μL:100 μL) extraction followed by one chloroform (600 μL) purification step.

Article Title: The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6
Article Snippet: After expression overnight at 22°C upon induction with 1 mM IPTG, the cultures were chemically lysed (0.4 M boric acid buffer, pH 8.0 containing 320 mM NaCl, 4 mM EDTA, 0.25% (w/v) lysozyme and 12.5 U ml−1 Benzonase® (Merck Millipore)) and the crude extracts were tested via ELISA for the presence of specifically binding antibody fragments. .. After three rounds of panning, the pool of Fab genes was isolated and inserted into E. coli expression vectors.

Bicinchoninic Acid Protein Assay:

Article Title: Protein Profiling Gastric Cancer and Neighboring Control Tissues Using High-Content Antibody Microarrays
Article Snippet: The material was lysed for 30 min on ice in 10 volumes of lysis buffer composed of 50 mM carbonate buffer (pH 8.5), 20% glycerol, 1.0 mM MgCl2 , 5.0 mM EDTA, 1.0 mM phenylmethanesulfonyl fluoride, 1.0 U/mL benzonase (Merck Biosciences, Schwalbach, Germany), Halt protease and phosphatase inhibitor mixture (Thermo Scientific, Bonn, Germany), 0.5% Nonidet P-40 substitute, 1.0% cholic acid, 0.25% n-dodecyl-maltoside (GenaXXon Bioscience, Ulm, Germany), and 0.5% amidosulfobetaine-14. .. The protein concentration was determined with the Bicinchoninic Acid Protein Assay Reagent (BCA) kit (Thermo Scientific) according to the manufacturer’s protocol.

Size-exclusion Chromatography:

Article Title: The C‐terminal cytosolic domain of the human zinc transporter ZnT8 and its diabetes risk variant
Article Snippet: .. To produce ZnT8c apo‐protein, 2 mm TCEP and 1 mm EDTA were added to the 10 mL fractions from size exclusion chromatography and then concentrated to ~ 0.5 mL using a 15 mL 3 kDa MWCO centrifugal concentrator (Merck Millipore). ..

Labeling:

Article Title: Protein Profiling Gastric Cancer and Neighboring Control Tissues Using High-Content Antibody Microarrays
Article Snippet: Paragraph title: 2.1. Protein Isolation and Labeling ... The material was lysed for 30 min on ice in 10 volumes of lysis buffer composed of 50 mM carbonate buffer (pH 8.5), 20% glycerol, 1.0 mM MgCl2 , 5.0 mM EDTA, 1.0 mM phenylmethanesulfonyl fluoride, 1.0 U/mL benzonase (Merck Biosciences, Schwalbach, Germany), Halt protease and phosphatase inhibitor mixture (Thermo Scientific, Bonn, Germany), 0.5% Nonidet P-40 substitute, 1.0% cholic acid, 0.25% n-dodecyl-maltoside (GenaXXon Bioscience, Ulm, Germany), and 0.5% amidosulfobetaine-14.

Purification:

Article Title: Evaluation of Potential Effects of NaCl and Sorbic Acid on Staphylococcal Enterotoxin A Formation
Article Snippet: For RNA extraction, each sample pellet was resuspended in 500 µL ice cold TES buffer, pH 7.5 (50 mM Tris (BDH Prolabo, VWR International, Stockholm, Sweden), 5 mM EDTA and 50 mM NaCl (Merck Millipore)) and the suspension was transferred to Precelly lysing kit tubes (VK 0.1) (Bertin Technologies, Montigny-le-Bretonneux, France). .. RNA was isolated using two steps of phenol-chloroform (600 μL:100 μL) extraction followed by one chloroform (600 μL) purification step.

Article Title: Cell-sized confinement controls generation and stability of a protein wave for spatiotemporal regulation in cells
Article Snippet: Paragraph title: Expression and purification of MinD and its mutant ... EL buffer was exchanged with storage buffer [50 mM HEPES-KOH (pH 7.6), 150 mM GluK, 10% glycerol, 0.1 mM EDTA] with 0.2 mM ADP-Mg by ultrafiltration using AmiconUltra-15 10 k and AmiconUltra-0.5 30 k filters (Merck Millipore).

Article Title: The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6
Article Snippet: After expression overnight at 22°C upon induction with 1 mM IPTG, the cultures were chemically lysed (0.4 M boric acid buffer, pH 8.0 containing 320 mM NaCl, 4 mM EDTA, 0.25% (w/v) lysozyme and 12.5 U ml−1 Benzonase® (Merck Millipore)) and the crude extracts were tested via ELISA for the presence of specifically binding antibody fragments. .. Colonies containing antibodies with unique CDR3 sequence were chosen for subsequent purification.

Article Title: The C‐terminal cytosolic domain of the human zinc transporter ZnT8 and its diabetes risk variant
Article Snippet: Paragraph title: Protein expression and purification ... To produce ZnT8c apo‐protein, 2 mm TCEP and 1 mm EDTA were added to the 10 mL fractions from size exclusion chromatography and then concentrated to ~ 0.5 mL using a 15 mL 3 kDa MWCO centrifugal concentrator (Merck Millipore).

Article Title: Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts
Article Snippet: Paragraph title: Carboxysome purification ... Cells were collected by centrifugation (6000 × g , 10 min) and resuspended in 25 mL TEMB buffer (5 mM Tris-HCl [pH 8.0], 1 mM EDTA, 10 mM MgCl2 , 20 mM NaHCO3 ) containing 0.55 M mannitol and 60 kU rLysozyme (Merck-Millipore, Bayswater, VIC, Australia).

Lysis:

Article Title: Protein Profiling Gastric Cancer and Neighboring Control Tissues Using High-Content Antibody Microarrays
Article Snippet: .. The material was lysed for 30 min on ice in 10 volumes of lysis buffer composed of 50 mM carbonate buffer (pH 8.5), 20% glycerol, 1.0 mM MgCl2 , 5.0 mM EDTA, 1.0 mM phenylmethanesulfonyl fluoride, 1.0 U/mL benzonase (Merck Biosciences, Schwalbach, Germany), Halt protease and phosphatase inhibitor mixture (Thermo Scientific, Bonn, Germany), 0.5% Nonidet P-40 substitute, 1.0% cholic acid, 0.25% n-dodecyl-maltoside (GenaXXon Bioscience, Ulm, Germany), and 0.5% amidosulfobetaine-14. .. Cell debris was pelleted by 20 min centrifugation at 4 °C and 13,000 rpm.

Titration:

Article Title: Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase
Article Snippet: .. All ITC measurements reported here were made in this buffer, using the following chemicals: glycine, ( > 99.7%, Merck, Darmstadt, Germany), ethylenediaminetetraacetic acid, EDTA ( > 99%, Merck, Darmstadt, Germany), and SDS > 99%, Fluka, Buchs, Switzerland) and a MCS-ITC (MicroCal, Northampton, MA) isothermal titration calorimeter. ..

SDS Page:

Article Title: The C‐terminal cytosolic domain of the human zinc transporter ZnT8 and its diabetes risk variant
Article Snippet: To produce ZnT8c apo‐protein, 2 mm TCEP and 1 mm EDTA were added to the 10 mL fractions from size exclusion chromatography and then concentrated to ~ 0.5 mL using a 15 mL 3 kDa MWCO centrifugal concentrator (Merck Millipore). .. Concentrated samples were analysed using 16% (w/v) Tris‐glycine SDS/PAGE (Thermo Fisher Scientific) .

RNA Extraction:

Article Title: Evaluation of Potential Effects of NaCl and Sorbic Acid on Staphylococcal Enterotoxin A Formation
Article Snippet: .. For RNA extraction, each sample pellet was resuspended in 500 µL ice cold TES buffer, pH 7.5 (50 mM Tris (BDH Prolabo, VWR International, Stockholm, Sweden), 5 mM EDTA and 50 mM NaCl (Merck Millipore)) and the suspension was transferred to Precelly lysing kit tubes (VK 0.1) (Bertin Technologies, Montigny-le-Bretonneux, France). .. Cells were disrupted in a Precellys 24 unit (Bertin Technologies), in a three cycle run of 60 s at 6500 rpm.

Recombinant:

Article Title: The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6
Article Snippet: The first round of washing was performed six times for 1 min, in the second round a more stringent washing was done eight times for 1 min 30 s, and in the third round the beads were washed eight times for 3 min with PBS buffer containing 0.05% (v/v) Tween 20 (PBST), followed in each round by one wash cycle in PBS for 2 min. To obtain species-specific antibodies against murine and human sclerostin, separate rounds of panning were performed on both recombinant proteins. .. After expression overnight at 22°C upon induction with 1 mM IPTG, the cultures were chemically lysed (0.4 M boric acid buffer, pH 8.0 containing 320 mM NaCl, 4 mM EDTA, 0.25% (w/v) lysozyme and 12.5 U ml−1 Benzonase® (Merck Millipore)) and the crude extracts were tested via ELISA for the presence of specifically binding antibody fragments.

In Vitro:

Article Title: Serine Proteases Degrade Airway Mucins in Cystic Fibrosis ▿Serine Proteases Degrade Airway Mucins in Cystic Fibrosis ▿ †
Article Snippet: Paragraph title: Mucin degradation in vitro and by proteases with and without protease inhibitors. ... Chemicals were purchased from Sigma (St. Louis, MO) (DFP [final concentration, 2 mM], PMSF [final concentration, 2 mM], TLCK [final concentration, 10 mM], EDTA [final concentration, 100 mM], and E64 [final concentration, 500 ng/ml]) or Merck Chemical (Nottingham, United Kingdom) (GM6001 [final concentration, 40 μM] and leupeptin [final concentration, 40 μM]).

Protein Binding:

Article Title: The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6
Article Snippet: In each panning round, the antibody phage library was incubated with biotinylated sclerostin protein, binding Fabs were captured via streptavidin-coated magnetic beads (Invitrogen) and phages were eluted with 25 mM DTT. .. After expression overnight at 22°C upon induction with 1 mM IPTG, the cultures were chemically lysed (0.4 M boric acid buffer, pH 8.0 containing 320 mM NaCl, 4 mM EDTA, 0.25% (w/v) lysozyme and 12.5 U ml−1 Benzonase® (Merck Millipore)) and the crude extracts were tested via ELISA for the presence of specifically binding antibody fragments.

Concentration Assay:

Article Title: Serine Proteases Degrade Airway Mucins in Cystic Fibrosis ▿Serine Proteases Degrade Airway Mucins in Cystic Fibrosis ▿ †
Article Snippet: .. Chemicals were purchased from Sigma (St. Louis, MO) (DFP [final concentration, 2 mM], PMSF [final concentration, 2 mM], TLCK [final concentration, 10 mM], EDTA [final concentration, 100 mM], and E64 [final concentration, 500 ng/ml]) or Merck Chemical (Nottingham, United Kingdom) (GM6001 [final concentration, 40 μM] and leupeptin [final concentration, 40 μM]). ..

Article Title: Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts
Article Snippet: Cells were collected by centrifugation (6000 × g , 10 min) and resuspended in 25 mL TEMB buffer (5 mM Tris-HCl [pH 8.0], 1 mM EDTA, 10 mM MgCl2 , 20 mM NaHCO3 ) containing 0.55 M mannitol and 60 kU rLysozyme (Merck-Millipore, Bayswater, VIC, Australia). .. IGEPAL CA-630 (Sigma-Aldrich, Castle Hill, NSW, Australia) was added to a final concentration of 1% (v /v ), and broken cells were mixed by inversion on a rotating shaker at 4 °C for 1 h. Cell debris and unbroken cells were removed by centrifugation at 3000 × g , 1 min, and the supernatant centrifuged at 40,000 × g for 20 min to generate a crude carboxysome pellet.

Article Title: Altered protease and antiprotease balance during a COPD exacerbation contributes to mucus obstruction
Article Snippet: .. DFP (final concentration 2 mM); PMSF (final concentration 2 mM); TLCK (final concentration 10 mM); EDTA (final concentration 100 mM); E64 (final concentration 500 ng/mL) or Merck Chemical (Nottingham, UK): GM6001 (final concentration 40 μM) and leupeptin (final concentration 40 μM) were used. .. Polyclonal anti-MUC5AC and anti-MUC5B antibodies were generated as previously described [ ].

Hydrophobic Interaction Chromatography:

Article Title: Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase
Article Snippet: HiC was extensively dialyzed at 5°C against 50 m M glycine, 2 m M EDTA, pH 10.0. .. All ITC measurements reported here were made in this buffer, using the following chemicals: glycine, ( > 99.7%, Merck, Darmstadt, Germany), ethylenediaminetetraacetic acid, EDTA ( > 99%, Merck, Darmstadt, Germany), and SDS > 99%, Fluka, Buchs, Switzerland) and a MCS-ITC (MicroCal, Northampton, MA) isothermal titration calorimeter.

Staining:

Article Title: Babesiosis due to the canine Babesia microti-like small piroplasm in dogs - first report from Portugal and possible vertical transmission
Article Snippet: .. Blood in EDTA was used to prepare thin glass-slide smears that were air-dried, fixed with methanol, stained with Hemacolor® (Merck, Germany) and then examined under light microscopy (magnification of 1000×) for the detection of possible piroplasms. .. Blood was also spotted onto individual papers (7.5 cm × 2.5 cm; GB 002 Schleicher and Schuell, Dassel, Germany) allowed to air-dry and stored at -20°C until further use.

Fluorescence In Situ Hybridization:

Article Title: The amyloid fold of Gad m 1 epitopes governs IgE binding
Article Snippet: .. Before use, the membranes were rinsed with ethanol, washed three times with TBS (25 mM Tris-HCl, pH 7.5, containing 137 mM NaCl and 2.7 mM KCl), and incubated in TBS containing 1% BSA (w/v) and 2 mM EDTA for 1 h. The membranes were then probed for 2 h with sera from patients who are allergic to fish (1/10 dilution) and the anti-amyloid fibril OC antibody (AB2286 Merck Millipore, 1/2,000 dilution), prepared in TBST (TBS containing 0.05% Tween-20) supplemented with 0.5% BSA (w/v) or with rGad m 1 chains (2.5 μM) in TBS. .. After extensive washes with TBST, a 30 min incubation with either horseradish peroxidase-labeled anti-human IgE (Abcam ab99806, 1/2,000 dilution), anti-human IgG4 (Abcam ab99823, 1/4,000 dilution), goat anti-rabbit IgG (Sigma, 1/5,000 dilution) or anti-6X His tag® antibody (Abcam ab18184, dilution 1/1,000) was performed.

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    Merck KGaA edta
    Dimerisation of the two human ZnT8 CTD variants. Representative ( n = 3) MST traces for dimerisation of ZnT8 CTD protein. Fluorescently labelled <t>apo‐ZnT8cR</t> (100 n m , magenta circles) was titrated (in the presence of 1 m m <t>EDTA</t> ) with unlabelled apo‐ZnT8cR protein (180 μ m –5.5 n m ), yielding a homodimerisation K d of 4.3 ± 1.3 μ m . Fluorescently labelled apo‐ZnT8cW (100 n m , teal triangles) was titrated (in the presence of 1 m m EDTA ) with unlabelled apo‐ZnT8cW protein (124 μ m –3.8 n m ), with a homodimerisation K d of 1.8 ± 0.1 μ m . There is a significant difference between the homodimerisation K d of each variant in the presence of EDTA ( n = 3, P = 0.034).
    Edta, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Dimerisation of the two human ZnT8 CTD variants. Representative ( n = 3) MST traces for dimerisation of ZnT8 CTD protein. Fluorescently labelled apo‐ZnT8cR (100 n m , magenta circles) was titrated (in the presence of 1 m m EDTA ) with unlabelled apo‐ZnT8cR protein (180 μ m –5.5 n m ), yielding a homodimerisation K d of 4.3 ± 1.3 μ m . Fluorescently labelled apo‐ZnT8cW (100 n m , teal triangles) was titrated (in the presence of 1 m m EDTA ) with unlabelled apo‐ZnT8cW protein (124 μ m –3.8 n m ), with a homodimerisation K d of 1.8 ± 0.1 μ m . There is a significant difference between the homodimerisation K d of each variant in the presence of EDTA ( n = 3, P = 0.034).

    Journal: The Febs Journal

    Article Title: The C‐terminal cytosolic domain of the human zinc transporter ZnT8 and its diabetes risk variant

    doi: 10.1111/febs.14402

    Figure Lengend Snippet: Dimerisation of the two human ZnT8 CTD variants. Representative ( n = 3) MST traces for dimerisation of ZnT8 CTD protein. Fluorescently labelled apo‐ZnT8cR (100 n m , magenta circles) was titrated (in the presence of 1 m m EDTA ) with unlabelled apo‐ZnT8cR protein (180 μ m –5.5 n m ), yielding a homodimerisation K d of 4.3 ± 1.3 μ m . Fluorescently labelled apo‐ZnT8cW (100 n m , teal triangles) was titrated (in the presence of 1 m m EDTA ) with unlabelled apo‐ZnT8cW protein (124 μ m –3.8 n m ), with a homodimerisation K d of 1.8 ± 0.1 μ m . There is a significant difference between the homodimerisation K d of each variant in the presence of EDTA ( n = 3, P = 0.034).

    Article Snippet: To produce ZnT8c apo‐protein, 2 mm TCEP and 1 mm EDTA were added to the 10 mL fractions from size exclusion chromatography and then concentrated to ~ 0.5 mL using a 15 mL 3 kDa MWCO centrifugal concentrator (Merck Millipore).

    Techniques: Microscale Thermophoresis, Variant Assay

    Serine proteases inhibit mucin degradation. Analysis of sputum MUC5AC and MUC5B by western blot after incubation at 37 °C over 24 h with or without of protease inhibitors. Sputum was obtained from a COPD subject 5–6 weeks after an acute exacerbation. Mucin concentration of the native control without incubation over 24 h was set to 100 %. a We used the serine protease inhibitors DFP, PMSF and TLCK, the metalloprotease inhibitors EDTA and GM6001 and the cysteine protease inhibitors leupeptin and E64. Analysis was performed in triplicate. b Incubation of COPD sputa (5–6 weeks after the onset) with A1-PI ( n = 4) and compared with control sputa

    Journal: Respiratory Research

    Article Title: Altered protease and antiprotease balance during a COPD exacerbation contributes to mucus obstruction

    doi: 10.1186/s12931-015-0247-x

    Figure Lengend Snippet: Serine proteases inhibit mucin degradation. Analysis of sputum MUC5AC and MUC5B by western blot after incubation at 37 °C over 24 h with or without of protease inhibitors. Sputum was obtained from a COPD subject 5–6 weeks after an acute exacerbation. Mucin concentration of the native control without incubation over 24 h was set to 100 %. a We used the serine protease inhibitors DFP, PMSF and TLCK, the metalloprotease inhibitors EDTA and GM6001 and the cysteine protease inhibitors leupeptin and E64. Analysis was performed in triplicate. b Incubation of COPD sputa (5–6 weeks after the onset) with A1-PI ( n = 4) and compared with control sputa

    Article Snippet: DFP (final concentration 2 mM); PMSF (final concentration 2 mM); TLCK (final concentration 10 mM); EDTA (final concentration 100 mM); E64 (final concentration 500 ng/mL) or Merck Chemical (Nottingham, UK): GM6001 (final concentration 40 μM) and leupeptin (final concentration 40 μM) were used.

    Techniques: Western Blot, Incubation, Concentration Assay

    Scanning electron micrographs (a) 1% oregano extract solution (OES) + 17% ethylenediaminetetraacetic acid (EDTA) (b) 2% OES + 17% EDTA (c) 5% OES + 17% EDTA

    Journal: European Journal of Dentistry

    Article Title: Antibacterial and smear layer removal capability of oregano extract solution

    doi: 10.4103/1305-7456.149633

    Figure Lengend Snippet: Scanning electron micrographs (a) 1% oregano extract solution (OES) + 17% ethylenediaminetetraacetic acid (EDTA) (b) 2% OES + 17% EDTA (c) 5% OES + 17% EDTA

    Article Snippet: The smear layer was removed in the first seven groups with 3 ml 17% ethylenediaminetetraacetic acid (EDTA) (Merck KGaA, Darmstadt, Germany) for 1-min, followed by 3 ml 5.25% NaOCl (Wizard, Ankara, Turkey) for 1-min, and then 5 ml distillate water for 1-min as outlined by Teixeira et al .

    Techniques:

    Scanning electron micrographs of 5.25 NaOCl + 17% ethylenediaminetetraacetic acid

    Journal: European Journal of Dentistry

    Article Title: Antibacterial and smear layer removal capability of oregano extract solution

    doi: 10.4103/1305-7456.149633

    Figure Lengend Snippet: Scanning electron micrographs of 5.25 NaOCl + 17% ethylenediaminetetraacetic acid

    Article Snippet: The smear layer was removed in the first seven groups with 3 ml 17% ethylenediaminetetraacetic acid (EDTA) (Merck KGaA, Darmstadt, Germany) for 1-min, followed by 3 ml 5.25% NaOCl (Wizard, Ankara, Turkey) for 1-min, and then 5 ml distillate water for 1-min as outlined by Teixeira et al .

    Techniques:

    Analysis of sputum MUC5AC and MUC5B by Western blotting after incubation at 37°C over 6 h in the presence or absence of proteases inhibitors. Sputum was obtained from a CF subject chronically infected by P. aeruginosa . The mucin concentration of the native control without incubation was set to 100%, and results are presented relative to this. We used the serine protease inhibitors DFP, PMSF, and TLCK, the metalloprotease inhibitors EDTA and GM6001, and the cysteine protease inhibitors leupeptin and E64.

    Journal: Infection and Immunity

    Article Title: Serine Proteases Degrade Airway Mucins in Cystic Fibrosis ▿Serine Proteases Degrade Airway Mucins in Cystic Fibrosis ▿ †

    doi: 10.1128/IAI.01252-10

    Figure Lengend Snippet: Analysis of sputum MUC5AC and MUC5B by Western blotting after incubation at 37°C over 6 h in the presence or absence of proteases inhibitors. Sputum was obtained from a CF subject chronically infected by P. aeruginosa . The mucin concentration of the native control without incubation was set to 100%, and results are presented relative to this. We used the serine protease inhibitors DFP, PMSF, and TLCK, the metalloprotease inhibitors EDTA and GM6001, and the cysteine protease inhibitors leupeptin and E64.

    Article Snippet: Chemicals were purchased from Sigma (St. Louis, MO) (DFP [final concentration, 2 mM], PMSF [final concentration, 2 mM], TLCK [final concentration, 10 mM], EDTA [final concentration, 100 mM], and E64 [final concentration, 500 ng/ml]) or Merck Chemical (Nottingham, United Kingdom) (GM6001 [final concentration, 40 μM] and leupeptin [final concentration, 40 μM]).

    Techniques: Western Blot, Incubation, Infection, Concentration Assay