edta free protease inhibitor cocktail  (Millipore)

 
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    Name:
    cOmplete EDTA free Protease Inhibitor Cocktail
    Description:
    cOmplete EDTA free Protease Inhibitor Tablets inhibit a broad spectrum of serine and cysteine proteases In contrast to other cOmplete tablets they do not contain EDTA no other chelating agents e g EGTA as well leaving the stability and the function of metal dependant proteins unaffected The affinity purification of Poly His tagged fusion proteins using IMAC Immobilized Metal Affinity Chromatography is also facilitated no dialysis necessary Due to the optimized composition of the tablets they show excellent inhibition of serine and cysteine proteases and are well suited for the protection of proteins isolated from animal tissues plants yeast and bacteria cOmplete EDTA free tablets contains both irreversible and reversible protease inhibitors Metallo and aspartic proteases are not inhibited cOmplete EDTA free Tablets are identical to cOmplete Tablets the only difference being that no EDTA or other chelating e g EGTA agents are included
    Catalog Number:
    04693132001
    Price:
    None
    Applications:
    cOmplete(TM), EDTA-free, efficiently inhibits serine and cysteine proteases in bacterial, yeast, plant, and animal cell extracts. Metallo- and aspartic proteases are not inhibited.cOmplete, EDTA-free Tablets, are used for the inhibition of proteolytic activity in large volumes (up to 50ml) in which EDTA may interfere with protein stability (e.g., metal-containing proteins) or subsequent assays. Since EDTA interferes with IMAC (Immobilized Metal Affinity Chromatography), cOmplete, EDTA-free is preferentially used in the isolation process of Poly-His-tagged fusion proteins.If it is necessary to inhibit proteolytic activity in a smaller volume (up to 10ml), we recommend to use cOmplete, Mini, EDTA-free.One cOmplete, EDTA-free tablet was added per 50 ml incubation solution. Proteolytic activity was determined with the Roche Universal Protease Substrate (casein, resorufin-labeled). When extractions or single-step isolations are necessary in the acid pH range, simply include pepstatin along with cOmplete, EDTA-free tablets to ensure aspartic (acid) protease inhibition. All experiments were performed at +15 to +25C.
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    Structured Review

    Millipore edta free protease inhibitor cocktail
    cOmplete EDTA free Protease Inhibitor Cocktail
    cOmplete EDTA free Protease Inhibitor Tablets inhibit a broad spectrum of serine and cysteine proteases In contrast to other cOmplete tablets they do not contain EDTA no other chelating agents e g EGTA as well leaving the stability and the function of metal dependant proteins unaffected The affinity purification of Poly His tagged fusion proteins using IMAC Immobilized Metal Affinity Chromatography is also facilitated no dialysis necessary Due to the optimized composition of the tablets they show excellent inhibition of serine and cysteine proteases and are well suited for the protection of proteins isolated from animal tissues plants yeast and bacteria cOmplete EDTA free tablets contains both irreversible and reversible protease inhibitors Metallo and aspartic proteases are not inhibited cOmplete EDTA free Tablets are identical to cOmplete Tablets the only difference being that no EDTA or other chelating e g EGTA agents are included
    https://www.bioz.com/result/edta free protease inhibitor cocktail/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    edta free protease inhibitor cocktail - by Bioz Stars, 2021-03
    99/100 stars

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    other:

    Article Title: Preparation, Functional Characterization, and NMR Studies of Human KCNE1, a Voltage-Gated Potassium Channel Accessory Subunit Associated With Deafness and Long QT Syndrome †
    Article Snippet: To the eluted protein solution EDTA and DTT were added to 2 mM and D2O was added to 10% (v/v).

    Protease Inhibitor:

    Article Title: Role of CovR phosphorylation in gene transcription in Streptococcus mutans
    Article Snippet: Cell lysates were prepared and care was taken to keep them chilled to minimize spontaneous dephosphorylation. .. The cells were first resuspended in 1.0 ml of cold 50 mM Tris pH 7.0 and 8 µl of Protease Inhibitor Cocktail Set III, EDTA-free (Calbiochem). .. Cell suspensions were then transferred to chilled Lysing Matrix B tubes (MP Biomedicals) and lysed with five consecutive runs of 30 s each at a speed of 6 m s−1 .

    Article Title: Mapping of Plasma Membrane Proteins Interacting With Arabidopsis thaliana Flotillin 2
    Article Snippet: .. Leaves were ground with a pestle and mortar in liquid nitrogen and further homogenized by sonication for 3 × 35 s (25 W) in 90 ml of extraction buffer (50 mM HEPES pH 7.5, 400 mM sucrose, 85 mM KCl, 100 mM MgCl2 .6H2 O, 0.02 mM ascorbic acid) containing cOmpleteTM EDTA-free Protease Inhibitor Cocktail according to the manufacturer’s instructions (Sigma Aldrich). .. The supernatant was filtered through Miracloth (Millipore) and centrifuged at 200,000 × g for 1 h at 4°C.

    Article Title: The protein kinase CK2 catalytic domain from Plasmodium falciparum: crystal structure, tyrosine kinase activity and inhibition
    Article Snippet: After overnight incubation, cells were harvested at 4000 × g for 10 minutes and re-suspended in the lysis buffer (100 mM Na Hepes, 500 mM NaCl, 10 mM Imidazole, 10% (v/v) Glycerol and 0.5 mM Tris (2-carboxyethyl) phosphine (TCEP) at pH 8.0. .. Pellets stored at −80 °C were thawed in an iced-water bath with 25 µl of EDTA-free protease inhibitor cocktail (Calbiochem) and 10 mg lysozyme added to the thawed cell re-suspension. .. Sonication was applied in cycles of 3 seconds pulses followed by 5 seconds delays at 30% amplitude for 5 minutes (Sonics Vibra-cell).

    Article Title: USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A
    Article Snippet: .. Cells were collected in lysis buffer (50 mM TRIS pH 8, 100 mM NaCl, 1 mM β-mercaptoethanol) with Complete EDTA-free protease inhibitor (Sigma) and lysed by sonication. .. Lysate was cleared by spinning down at 21,000 × g and loaded on TALON beads (Clontech Laboratories, Inc.).

    Article Title: Protective effect of stromal Dickkopf-3 in prostate cancer: opposing roles for TGFBI and ECM-1
    Article Snippet: In some experiments, media were changed to serum-free with or without 10 ng/ml TGF-β (R & D Systems) for 24 h. CM were centrifuged at 500 × g for 5 min and supernatants added to an equal volume of 2× Laemmli buffer (Sigma). .. Cells were lysed on ice in RIPA lysis buffer (0.5% Na deoxycholate, 1% Triton X-100, 20 mM Tris pH 8.0, 0.1% SDS, 100 mM NaCl, 50 mM NaF, 1 mM EDTA), cOmplete™ EDTA-free Protease Inhibitor Cocktail (Sigma) and PhosSTOP Phosphatase Inhibitor Cocktail (Sigma)). .. Lysates were incubated for 15 min on ice, centrifuged at 15,000× g for 15 min at 4 °C and added to an equal volume of 2× Laemmli buffer.

    Article Title: An Internally Controlled Quantitative Target Occupancy Assay for Covalent Inhibitors
    Article Snippet: .. Frozen resected or needle biopsy tumor samples were homogenized using a Precellys 24 homogenizer (Bertin Instruments; Montigny-le-Bretonneux, France) using 2.0 mm Zirconia Beads (BioSpec Products; Bartlesville, OK) and solubilized in ice-cold lysis buffer (Pierce™ IP Lysis Buffer; Thermo Fisher Scientific) supplemented with cOmplete EDTA-free protease inhibitor cocktail Tablet (MilliporeSigma). .. Tumor lysates were centrifuged for 5 minutes at 17,000 x g to remove insoluble debris, supernatant recovered and protein concentration measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).

    Article Title: Structures of chaperone-substrate complexes docked onto the export gate in a type III secretion system
    Article Snippet: All mutants were constructed by site-directed mutagenesis using PfuTurbo high-fidelity DNA polymerase (Agilent). .. Unlabeled proteins were expressed in BL21(DE3) cells grown in Luria-Bertani (LB) medium in the presence of ampicillin (100 μg ml−1 ) at 37 ℃, and protein expression was induced at 18 ℃ with 0.4 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at OD600 ≈ 0.5 for ~48 h. Cells were harvested at OD600 ≈ 1.5 and were suspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% EDTA-free protease inhibitor cocktail (Sigma-Aldrich), and 20 mM imidazole. .. Cells were disrupted by a high-pressure homogenizer and centrifuged at 20,000 r.p.m. for 1 h. Proteins were purified using Ni Sepharose 6 Fast Flow resin (GE Healthcare), followed by tag removal by TEV protease at 4 ℃ for 12–20 h and gel filtration using Superdex 75 16/60.

    Sonication:

    Article Title: Mapping of Plasma Membrane Proteins Interacting With Arabidopsis thaliana Flotillin 2
    Article Snippet: .. Leaves were ground with a pestle and mortar in liquid nitrogen and further homogenized by sonication for 3 × 35 s (25 W) in 90 ml of extraction buffer (50 mM HEPES pH 7.5, 400 mM sucrose, 85 mM KCl, 100 mM MgCl2 .6H2 O, 0.02 mM ascorbic acid) containing cOmpleteTM EDTA-free Protease Inhibitor Cocktail according to the manufacturer’s instructions (Sigma Aldrich). .. The supernatant was filtered through Miracloth (Millipore) and centrifuged at 200,000 × g for 1 h at 4°C.

    Article Title: USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A
    Article Snippet: .. Cells were collected in lysis buffer (50 mM TRIS pH 8, 100 mM NaCl, 1 mM β-mercaptoethanol) with Complete EDTA-free protease inhibitor (Sigma) and lysed by sonication. .. Lysate was cleared by spinning down at 21,000 × g and loaded on TALON beads (Clontech Laboratories, Inc.).

    Lysis:

    Article Title: USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A
    Article Snippet: .. Cells were collected in lysis buffer (50 mM TRIS pH 8, 100 mM NaCl, 1 mM β-mercaptoethanol) with Complete EDTA-free protease inhibitor (Sigma) and lysed by sonication. .. Lysate was cleared by spinning down at 21,000 × g and loaded on TALON beads (Clontech Laboratories, Inc.).

    Article Title: Protective effect of stromal Dickkopf-3 in prostate cancer: opposing roles for TGFBI and ECM-1
    Article Snippet: In some experiments, media were changed to serum-free with or without 10 ng/ml TGF-β (R & D Systems) for 24 h. CM were centrifuged at 500 × g for 5 min and supernatants added to an equal volume of 2× Laemmli buffer (Sigma). .. Cells were lysed on ice in RIPA lysis buffer (0.5% Na deoxycholate, 1% Triton X-100, 20 mM Tris pH 8.0, 0.1% SDS, 100 mM NaCl, 50 mM NaF, 1 mM EDTA), cOmplete™ EDTA-free Protease Inhibitor Cocktail (Sigma) and PhosSTOP Phosphatase Inhibitor Cocktail (Sigma)). .. Lysates were incubated for 15 min on ice, centrifuged at 15,000× g for 15 min at 4 °C and added to an equal volume of 2× Laemmli buffer.

    Article Title: An Internally Controlled Quantitative Target Occupancy Assay for Covalent Inhibitors
    Article Snippet: .. Frozen resected or needle biopsy tumor samples were homogenized using a Precellys 24 homogenizer (Bertin Instruments; Montigny-le-Bretonneux, France) using 2.0 mm Zirconia Beads (BioSpec Products; Bartlesville, OK) and solubilized in ice-cold lysis buffer (Pierce™ IP Lysis Buffer; Thermo Fisher Scientific) supplemented with cOmplete EDTA-free protease inhibitor cocktail Tablet (MilliporeSigma). .. Tumor lysates were centrifuged for 5 minutes at 17,000 x g to remove insoluble debris, supernatant recovered and protein concentration measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).

    Article Title: Structures of chaperone-substrate complexes docked onto the export gate in a type III secretion system
    Article Snippet: All mutants were constructed by site-directed mutagenesis using PfuTurbo high-fidelity DNA polymerase (Agilent). .. Unlabeled proteins were expressed in BL21(DE3) cells grown in Luria-Bertani (LB) medium in the presence of ampicillin (100 μg ml−1 ) at 37 ℃, and protein expression was induced at 18 ℃ with 0.4 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at OD600 ≈ 0.5 for ~48 h. Cells were harvested at OD600 ≈ 1.5 and were suspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% EDTA-free protease inhibitor cocktail (Sigma-Aldrich), and 20 mM imidazole. .. Cells were disrupted by a high-pressure homogenizer and centrifuged at 20,000 r.p.m. for 1 h. Proteins were purified using Ni Sepharose 6 Fast Flow resin (GE Healthcare), followed by tag removal by TEV protease at 4 ℃ for 12–20 h and gel filtration using Superdex 75 16/60.

    Expressing:

    Article Title: Structures of chaperone-substrate complexes docked onto the export gate in a type III secretion system
    Article Snippet: All mutants were constructed by site-directed mutagenesis using PfuTurbo high-fidelity DNA polymerase (Agilent). .. Unlabeled proteins were expressed in BL21(DE3) cells grown in Luria-Bertani (LB) medium in the presence of ampicillin (100 μg ml−1 ) at 37 ℃, and protein expression was induced at 18 ℃ with 0.4 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at OD600 ≈ 0.5 for ~48 h. Cells were harvested at OD600 ≈ 1.5 and were suspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% EDTA-free protease inhibitor cocktail (Sigma-Aldrich), and 20 mM imidazole. .. Cells were disrupted by a high-pressure homogenizer and centrifuged at 20,000 r.p.m. for 1 h. Proteins were purified using Ni Sepharose 6 Fast Flow resin (GE Healthcare), followed by tag removal by TEV protease at 4 ℃ for 12–20 h and gel filtration using Superdex 75 16/60.

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  • 99
    Millipore protease inhibitors edta
    Streptococcal protease responsible for PAR-1 cleavage. ( A ) GAS supernatants pre-incubated with either the metalloprotease inhibitor <t>EDTA,</t> the serine protein inhibitors <t>PMSF</t> and benzamidine (BAM), the cysteine protease inhibitor E64 or buffer were added onto 293T cells transiently over-expressing alkaline phosphatase-tagged PAR-1. Released alkaline phosphatase activity was quantified in the supernatants. ( B ) Cells as described in (A) were incubated with supernatants from exponential and stationary GAS to analyse their efficiency in cleaving AP-PAR-1 reporter constructs. ( C ) SpeB proteolytic activity was analysed by Bz-Pro-Phe-Arg-Nan cleavage in the exponential and stationary cultures GAS samples used in (B). Commercial SpeB (13.3 µg/ml) served as a positive control. ( D ) AP-PAR-1 reporter constructs were incubated with overnight cultures of non pre-treated wild type GAS, GAS preincubated with cysteine protease inhibitor E64 or the isogenic speB deficient GAS (GAS∆ speB ). In addition supernatants from GAS∆ speB were complemented with commercial SpeB (200nM) and cleavage of AP-PAR-1 reporter construct was carried out. Thrombin (IIa, 1nM) served as positive control. Experiments were repeated at least 3 times with N=9, data presented as mean +/- SEM, * P
    Protease Inhibitors Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protease inhibitors edta/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    protease inhibitors edta - by Bioz Stars, 2021-03
    99/100 stars
      Buy from Supplier

    99
    Millipore edta free
    Differential binding of wild-type and mutant CovR. Biotinylated promoter regions of gbpC and SMU.1882 were immobilized on streptavidin biosensors and then exposed to 0.5 µM CovR, CovR D53A and CovR D53E proteins purified from E. coli as described in the text. The reactions were carried out in binding buffer [20 mM <t>Tris,</t> 100 mM NaCl, 0.01 mM DTT, 5 % glycerol (v/v), 1 mM <t>EDTA,</t> 0.01 mg ml −1 BSA, 5 mM MgCl 2 and 10 µg ml −1 poly (dI-dC) at pH 7.5] for a period of 5 min to allow association followed by 2 min exposure to binding buffer to allow dissociation.
    Edta Free, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/edta free/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    edta free - by Bioz Stars, 2021-03
    99/100 stars
      Buy from Supplier

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    Streptococcal protease responsible for PAR-1 cleavage. ( A ) GAS supernatants pre-incubated with either the metalloprotease inhibitor EDTA, the serine protein inhibitors PMSF and benzamidine (BAM), the cysteine protease inhibitor E64 or buffer were added onto 293T cells transiently over-expressing alkaline phosphatase-tagged PAR-1. Released alkaline phosphatase activity was quantified in the supernatants. ( B ) Cells as described in (A) were incubated with supernatants from exponential and stationary GAS to analyse their efficiency in cleaving AP-PAR-1 reporter constructs. ( C ) SpeB proteolytic activity was analysed by Bz-Pro-Phe-Arg-Nan cleavage in the exponential and stationary cultures GAS samples used in (B). Commercial SpeB (13.3 µg/ml) served as a positive control. ( D ) AP-PAR-1 reporter constructs were incubated with overnight cultures of non pre-treated wild type GAS, GAS preincubated with cysteine protease inhibitor E64 or the isogenic speB deficient GAS (GAS∆ speB ). In addition supernatants from GAS∆ speB were complemented with commercial SpeB (200nM) and cleavage of AP-PAR-1 reporter construct was carried out. Thrombin (IIa, 1nM) served as positive control. Experiments were repeated at least 3 times with N=9, data presented as mean +/- SEM, * P

    Journal: PLoS ONE

    Article Title: Streptococcal SpeB Cleaved PAR-1 Suppresses ERK Phosphorylation and Blunts Thrombin-Induced Platelet Aggregation

    doi: 10.1371/journal.pone.0081298

    Figure Lengend Snippet: Streptococcal protease responsible for PAR-1 cleavage. ( A ) GAS supernatants pre-incubated with either the metalloprotease inhibitor EDTA, the serine protein inhibitors PMSF and benzamidine (BAM), the cysteine protease inhibitor E64 or buffer were added onto 293T cells transiently over-expressing alkaline phosphatase-tagged PAR-1. Released alkaline phosphatase activity was quantified in the supernatants. ( B ) Cells as described in (A) were incubated with supernatants from exponential and stationary GAS to analyse their efficiency in cleaving AP-PAR-1 reporter constructs. ( C ) SpeB proteolytic activity was analysed by Bz-Pro-Phe-Arg-Nan cleavage in the exponential and stationary cultures GAS samples used in (B). Commercial SpeB (13.3 µg/ml) served as a positive control. ( D ) AP-PAR-1 reporter constructs were incubated with overnight cultures of non pre-treated wild type GAS, GAS preincubated with cysteine protease inhibitor E64 or the isogenic speB deficient GAS (GAS∆ speB ). In addition supernatants from GAS∆ speB were complemented with commercial SpeB (200nM) and cleavage of AP-PAR-1 reporter construct was carried out. Thrombin (IIa, 1nM) served as positive control. Experiments were repeated at least 3 times with N=9, data presented as mean +/- SEM, * P

    Article Snippet: Protease inhibitors EDTA, PMSF, benzamidin and E64 were from Sigma-Aldrich Chemie GmbH (Buchs, CH).

    Techniques: Incubation, Protease Inhibitor, Expressing, Activity Assay, Construct, Positive Control

    Effect of oral treatment with TreXTAM on levels of transforming growth factor in colon. Treated rats ( n =3) received TreXTAM [60 mg/kg encapsulated transforming growth factor (TGF) and 30 mg/kg encapsulated all trans retinoic acid (ATRA)] three times a week for four weeks. Colons of these animals were taken 4 h after the final dose, along with tissues from age and sex matched untreated animals. All tissues were frozen at -20 o C and stored until used. Tissues were then thawed and homogenized using a glass tube with the pestle insert, in the presence of EDTA-free SIGMAFAST™ Protease Inhibitor Cocktail Tablets (used as per manufacturer’s instructions). Levels of TGF in lysates were determined by ELISA according to manufacturer’s instructions but without acid activation. (Quantikine, R D Systems, Minneapolis, MN, United States). Total protein concentration was determined by BCA Protein Assay- Pierce (Thermo Fisher Cat# 23227) Data are expressed as pg/100 μg protein ± standard deviation. TGFβ: Transforming growth factor .

    Journal: World Journal of Gastrointestinal Pharmacology and Therapeutics

    Article Title: Oral encapsulated transforming growth factor β1 reduces endogenous levels: Effect on inflammatory bowel disease

    doi: 10.4292/wjgpt.v11.i5.79

    Figure Lengend Snippet: Effect of oral treatment with TreXTAM on levels of transforming growth factor in colon. Treated rats ( n =3) received TreXTAM [60 mg/kg encapsulated transforming growth factor (TGF) and 30 mg/kg encapsulated all trans retinoic acid (ATRA)] three times a week for four weeks. Colons of these animals were taken 4 h after the final dose, along with tissues from age and sex matched untreated animals. All tissues were frozen at -20 o C and stored until used. Tissues were then thawed and homogenized using a glass tube with the pestle insert, in the presence of EDTA-free SIGMAFAST™ Protease Inhibitor Cocktail Tablets (used as per manufacturer’s instructions). Levels of TGF in lysates were determined by ELISA according to manufacturer’s instructions but without acid activation. (Quantikine, R D Systems, Minneapolis, MN, United States). Total protein concentration was determined by BCA Protein Assay- Pierce (Thermo Fisher Cat# 23227) Data are expressed as pg/100 μg protein ± standard deviation. TGFβ: Transforming growth factor .

    Article Snippet: Tissues were then thawed and homogenized using a glass tube with the pestle insert, in the presence of EDTA-free SIGMAFAST™ Protease Inhibitor Cocktail Tablets (Sigma-Aldrich) used as per manufacturer’s instructions.

    Techniques: Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Activation Assay, Protein Concentration, Bicinchoninic Acid Protein Assay, Standard Deviation

    Differential binding of wild-type and mutant CovR. Biotinylated promoter regions of gbpC and SMU.1882 were immobilized on streptavidin biosensors and then exposed to 0.5 µM CovR, CovR D53A and CovR D53E proteins purified from E. coli as described in the text. The reactions were carried out in binding buffer [20 mM Tris, 100 mM NaCl, 0.01 mM DTT, 5 % glycerol (v/v), 1 mM EDTA, 0.01 mg ml −1 BSA, 5 mM MgCl 2 and 10 µg ml −1 poly (dI-dC) at pH 7.5] for a period of 5 min to allow association followed by 2 min exposure to binding buffer to allow dissociation.

    Journal: Microbiology

    Article Title: Role of CovR phosphorylation in gene transcription in Streptococcus mutans

    doi: 10.1099/mic.0.000641

    Figure Lengend Snippet: Differential binding of wild-type and mutant CovR. Biotinylated promoter regions of gbpC and SMU.1882 were immobilized on streptavidin biosensors and then exposed to 0.5 µM CovR, CovR D53A and CovR D53E proteins purified from E. coli as described in the text. The reactions were carried out in binding buffer [20 mM Tris, 100 mM NaCl, 0.01 mM DTT, 5 % glycerol (v/v), 1 mM EDTA, 0.01 mg ml −1 BSA, 5 mM MgCl 2 and 10 µg ml −1 poly (dI-dC) at pH 7.5] for a period of 5 min to allow association followed by 2 min exposure to binding buffer to allow dissociation.

    Article Snippet: The cells were first resuspended in 1.0 ml of cold 50 mM Tris pH 7.0 and 8 µl of Protease Inhibitor Cocktail Set III, EDTA-free (Calbiochem).

    Techniques: Binding Assay, Mutagenesis, Purification

    2-D 600 MHz 1 H- 15 N TROSY NMR spectra of purified U- 15 N-His 6 -KCNE1 at 1−1.5 mM in different detergent micelles. Samples contained 250 mM imidazole, 2mM DTT, and 2 mM EDTA, pH 6.0. All the spectra were acquired with 256 × 1024 complex points

    Journal:

    Article Title: Preparation, Functional Characterization, and NMR Studies of Human KCNE1, a Voltage-Gated Potassium Channel Accessory Subunit Associated With Deafness and Long QT Syndrome †

    doi: 10.1021/bi700705j

    Figure Lengend Snippet: 2-D 600 MHz 1 H- 15 N TROSY NMR spectra of purified U- 15 N-His 6 -KCNE1 at 1−1.5 mM in different detergent micelles. Samples contained 250 mM imidazole, 2mM DTT, and 2 mM EDTA, pH 6.0. All the spectra were acquired with 256 × 1024 complex points

    Article Snippet: To the eluted protein solution EDTA and DTT were added to 2 mM and D2O was added to 10% (v/v).

    Techniques: Nuclear Magnetic Resonance, Purification

    1 H- 15 N TROSY spectrum of uniformly- 2 H, 13 C, 15 N-labeled KCNE1 in 4% LMPG, 10% D2O, 250 mM imidazole/acetic acid, 2 mM DTT, and 2 mM EDTA, pH 6.0. The spectrum was recorded at 40°C on a 900 MHz spectrometer with 256 complex points in t1, 1024 complex

    Journal:

    Article Title: Preparation, Functional Characterization, and NMR Studies of Human KCNE1, a Voltage-Gated Potassium Channel Accessory Subunit Associated With Deafness and Long QT Syndrome †

    doi: 10.1021/bi700705j

    Figure Lengend Snippet: 1 H- 15 N TROSY spectrum of uniformly- 2 H, 13 C, 15 N-labeled KCNE1 in 4% LMPG, 10% D2O, 250 mM imidazole/acetic acid, 2 mM DTT, and 2 mM EDTA, pH 6.0. The spectrum was recorded at 40°C on a 900 MHz spectrometer with 256 complex points in t1, 1024 complex

    Article Snippet: To the eluted protein solution EDTA and DTT were added to 2 mM and D2O was added to 10% (v/v).

    Techniques: Labeling