halt protease  (Thermo Fisher)


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    Name:
    Halt Protease Inhibitor Cocktail
    Description:
    Thermo Scientific Halt Protease Inhibitor Cocktail EDTA free 100X are ready to use concentrated stock solutions of protease inhibitors for addition to samples to prevent proteolytic degradation during cell lysis and protein extraction Features of Halt Protease Inhibitor Single Use Cocktail EDTA free 100X • Convenient the refrigerator stable 100X liquid format is more effective and easier to use than individual inhibitors or other formats just pipette exactly the amount you need • No proprietary ingredients fully disclosed formulation contains optimized concentrations of six broad spectrum protease inhibitors AEBSF aprotinin bestatin E 64 leupeptin and pepstatin A stabilized in high quality dimethylsulfoxide DMSO • Multiple package sizes choose among several package sizes including single use microtubes that keep protease inhibitors fresh and reduce the risk of reagent contamination • EDTA free formulations ensures compatibility with isoelectric focusing • Lysis buffer compatible use with Thermo Scientific Pierce Cell Lysis Buffers or nearly any other commercial or homemade detergent based lysis reagent Applications • Protection of intact active cellular proteins from degradation by endogenous proteases • Inhibition of protease activity from lysates produced by a variety of methods including Thermo Scientific Pierce Cell Lysis Reagents other commercially formulated cell or tissue lysis products sonication French press blender homogenization or repetitive freeze thaw cycling • Screening of extracts for proteolytic activity • The study of proteolysis in the regulation of cellular processes Add 10 µL of concentrated cocktail per 1 mL of lysis buffer to ensure complete protection of the resulting protein extract The single use format is ideal for small scale cell lysis experiments where each 100 µL vial provides just what is needed for a 10 mL sample and there is no risk of contaminating or degrading remaining aliquots of cocktail Each microtube in the strip is heat sealed with a foil that can be easily punctured by a pipette tip for trouble free access to a precise amount of Halt Protease Inhibitor Cocktail needed for each experiment Proteases make up a large category of enzymes including endopeptidases and exopeptidases which catalyze the hydrolytic breakdown of proteins into peptides or amino acids in a wide variety of cell and tissue types Proteases are regulated and compartmentalized in living cells but cell lysis releases and mixes them with other extracted proteins Their activity can thus lead to the loss of a large number of valuable proteins adversely affecting downstream applications For this reason it is common practice to spike cell lysis buffers or cell extracts with a cocktails of compounds known to inactivate or inhibit proteases Halt Protease Inhibitor Cocktails effectively inhibit serine proteases cysteine proteases aspartic acid proteases and aminopeptidases that are typically present in cellular lysate samples Regular and EDTA free formulations provide for inhibition of metalloproteases and compatibility with 2D electrophoresis respectively Related Products Halt Protease Inhibitor Single Use Cocktail 100X
    Catalog Number:
    78425
    Price:
    None
    Category:
    Lab Reagents and Chemicals
    Applications:
    Cell Lysis & Fractionation|Protease and Phosphatase Inhibition|Protein Biology|Protein Purification & Isolation
    Buy from Supplier


    Structured Review

    Thermo Fisher halt protease
    Thermo Scientific Halt Protease Inhibitor Cocktail EDTA free 100X are ready to use concentrated stock solutions of protease inhibitors for addition to samples to prevent proteolytic degradation during cell lysis and protein extraction Features of Halt Protease Inhibitor Single Use Cocktail EDTA free 100X • Convenient the refrigerator stable 100X liquid format is more effective and easier to use than individual inhibitors or other formats just pipette exactly the amount you need • No proprietary ingredients fully disclosed formulation contains optimized concentrations of six broad spectrum protease inhibitors AEBSF aprotinin bestatin E 64 leupeptin and pepstatin A stabilized in high quality dimethylsulfoxide DMSO • Multiple package sizes choose among several package sizes including single use microtubes that keep protease inhibitors fresh and reduce the risk of reagent contamination • EDTA free formulations ensures compatibility with isoelectric focusing • Lysis buffer compatible use with Thermo Scientific Pierce Cell Lysis Buffers or nearly any other commercial or homemade detergent based lysis reagent Applications • Protection of intact active cellular proteins from degradation by endogenous proteases • Inhibition of protease activity from lysates produced by a variety of methods including Thermo Scientific Pierce Cell Lysis Reagents other commercially formulated cell or tissue lysis products sonication French press blender homogenization or repetitive freeze thaw cycling • Screening of extracts for proteolytic activity • The study of proteolysis in the regulation of cellular processes Add 10 µL of concentrated cocktail per 1 mL of lysis buffer to ensure complete protection of the resulting protein extract The single use format is ideal for small scale cell lysis experiments where each 100 µL vial provides just what is needed for a 10 mL sample and there is no risk of contaminating or degrading remaining aliquots of cocktail Each microtube in the strip is heat sealed with a foil that can be easily punctured by a pipette tip for trouble free access to a precise amount of Halt Protease Inhibitor Cocktail needed for each experiment Proteases make up a large category of enzymes including endopeptidases and exopeptidases which catalyze the hydrolytic breakdown of proteins into peptides or amino acids in a wide variety of cell and tissue types Proteases are regulated and compartmentalized in living cells but cell lysis releases and mixes them with other extracted proteins Their activity can thus lead to the loss of a large number of valuable proteins adversely affecting downstream applications For this reason it is common practice to spike cell lysis buffers or cell extracts with a cocktails of compounds known to inactivate or inhibit proteases Halt Protease Inhibitor Cocktails effectively inhibit serine proteases cysteine proteases aspartic acid proteases and aminopeptidases that are typically present in cellular lysate samples Regular and EDTA free formulations provide for inhibition of metalloproteases and compatibility with 2D electrophoresis respectively Related Products Halt Protease Inhibitor Single Use Cocktail 100X
    https://www.bioz.com/result/halt protease/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    halt protease - by Bioz Stars, 2021-03
    86/100 stars

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    Related Articles

    Protease Inhibitor:

    Article Title: Characterization of the Canine Anthracycline-Metabolizing Enzyme Carbonyl Reductase 1 (cbr1) and the Functional Isoform cbr1 V218
    Article Snippet: Bacterial cell pellets were harvested by centrifugation and stored at –80°C until further processing. .. Bacterial pellets were reconstituted and lysed in 3 ml of B-PER reagent (Thermo Fisher Scientific, Rockford, IL) supplemented with lysozyme, DNase I, and Halt Protease Inhibitor Cocktail, EDTA-free (Thermo Fisher Scientific) for 45 minutes at 4°C with slight agitation. .. His-tagged recombinant canine cbr1 proteins were isolated and purified using HisPur Colbalt Spin Columns (Thermo Fisher Scientific) following the manufacturer’s instructions.

    Article Title: The Redundancy of Peptidoglycan Carboxypeptidases Ensures Robust Cell Shape Maintenance in Escherichia coli
    Article Snippet: Cell growth was monitored until cultures reached an optical density at 600 nm (OD600 ) of 0.7 to 0.8, at which time the cells were harvested by centrifugation at 10,000 rpm for 10 min at 4°C, in a Sorvall benchtop centrifuge. .. The cell pellet was resuspended in 10 ml of filter-sterilized 25 mM citric acid-sodium phosphate buffer (pH 5.0 or pH 7.5, as required) containing Halt protease inhibitor (Pierce) (10 µl Halt for every 10 ml of resuspended sample) and DNase (4 µg/ml final concentration). .. Cells were lysed by using a low-volume, high-shear Microfluidizer LV1 cell disruptor (Microfluidics, Westwood, MA), and cell debris was removed by centrifugation at 10,000 rpm for 10 min at 4°C.

    Article Title: Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella
    Article Snippet: Quenched fluorescence protease activity assays Stock solutions (1.0 mM) of synthetic quenched fluorescence (QF) peptide substrates (GenScript Inc.) were dissolved in DMSO and working stocks (100 μM) were prepared using the molar extinction coefficient of the conjugated quencher, (2,4)-dinitrophenyl, of 6.985 cm–1 mM–1 at 400 nm. .. Purified recombinant FliA(H)-hypervariable region protease was incubated at a final concentration of 0.5 µM with 10 µM QF-peptide substrate in 100 µl of 150 mM NaCl, 10 mM CaCl2 , 50 mM HEPES, pH 7.5, in the presence of HALT protease inhibitor cocktail (Life Technologies) plus/minus 20 mM EDTA at 37 °C. .. Three different quenched fluorescent peptides (PLG↓L), Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg; ALG↓L, Mca-Ala-Leu-Gly-Leu-Dpa-Ala-Arg; and PLG↓V, Mca-Pro-Leu-Gly-Val-Dpa-Ala-Arg) were tested using a multi-wavelength fluorescence scanner (POLARstar OPTIMA, BMG Lab technologies).

    Article Title: Vasopressin type 1A receptor deletion enhances cardiac contractility, β-adrenergic receptor sensitivity and acute cardiac injury-induced dysfunction
    Article Snippet: A Tecan M1000 multimode plate reader was used to read the absorbance of the samples at 405 nm and Prism 5.0 software (GraphPad Software) was used to calculate the concentration of cAMP in the samples via interpolation from the linear regression of the standard curve. .. Left ventricular samples were homogenized in lysis buffer containing 20 mM Tris (pH 7.4), 137 mM NaCl, 10 % glycerol, 1 mM EDTA, 1 % NP-40, 10 mM NaF (Fisher Scientific), 1× HALT protease inhibitor cocktail (Thermo Scientific) and phosphatase inhibitor cocktail set IV (Calbiochem). .. Lysates were run on SDS/15 % PAGE gels and transferred to Immobilon-PSQ polyvinylidene fluoride 0.2 mm pore size membranes (Millipore), which were then incubated with anti-Phospho-Ser-16 phospholamban (PLB) rabbit pAb (1:5000, Badrilla), anti-PLB mouse mAb (1:1000, Badrilla), anti-Phospho-Ser-23/24 cTnI rabbit pAb (1:1000, Cell Signaling Technology) or anti-cTnI rabbit pAb (1:1000, Cell Signaling Technology).

    Article Title: A System for Creating Stable Cell Lines that Express a Gene of Interest from a Bidirectional and Regulatable Herpes Simplex Virus Type 1 Promoter
    Article Snippet: .. Western immunoblotCells were removed from plates with trypsin, washed with PBS and proteins extracted using M-PER (Thermo Scientific) supplemented with Halt Protease inhibitor cocktail (Thermo Scientific). .. After a 30-minute incubation on ice, samples were centrifuged (14,000 rpm/30 min/4°C), supernatants collected and protein concentrations determined by Pierce BCA Protein Assay (Thermo Scientific).

    Article Title: Expression and characterization of a codon-optimized alkaline-stable carbonic anhydrase from Aliivibrio salmonicida for CO2 sequestration applications.
    Article Snippet: The CO < sub > 2 < /sub > mineralization process, accelerated by carbonic anhydrase (CA) was proposed for the efficient capture and storage of CO < sub > 2 < /sub > , the accumulation of which in the atmosphere is the main cause of global warming. .. The CO < sub > 2 < /sub > mineralization process, accelerated by carbonic anhydrase (CA) was proposed for the efficient capture and storage of CO < sub > 2 < /sub > , the accumulation of which in the atmosphere is the main cause of global warming. .. The CO < sub > 2 < /sub > mineralization process, accelerated by carbonic anhydrase (CA) was proposed for the efficient capture and storage of CO < sub > 2 < /sub > , the accumulation of which in the atmosphere is the main cause of global warming.

    Concentration Assay:

    Article Title: The Redundancy of Peptidoglycan Carboxypeptidases Ensures Robust Cell Shape Maintenance in Escherichia coli
    Article Snippet: Cell growth was monitored until cultures reached an optical density at 600 nm (OD600 ) of 0.7 to 0.8, at which time the cells were harvested by centrifugation at 10,000 rpm for 10 min at 4°C, in a Sorvall benchtop centrifuge. .. The cell pellet was resuspended in 10 ml of filter-sterilized 25 mM citric acid-sodium phosphate buffer (pH 5.0 or pH 7.5, as required) containing Halt protease inhibitor (Pierce) (10 µl Halt for every 10 ml of resuspended sample) and DNase (4 µg/ml final concentration). .. Cells were lysed by using a low-volume, high-shear Microfluidizer LV1 cell disruptor (Microfluidics, Westwood, MA), and cell debris was removed by centrifugation at 10,000 rpm for 10 min at 4°C.

    Article Title: Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella
    Article Snippet: Quenched fluorescence protease activity assays Stock solutions (1.0 mM) of synthetic quenched fluorescence (QF) peptide substrates (GenScript Inc.) were dissolved in DMSO and working stocks (100 μM) were prepared using the molar extinction coefficient of the conjugated quencher, (2,4)-dinitrophenyl, of 6.985 cm–1 mM–1 at 400 nm. .. Purified recombinant FliA(H)-hypervariable region protease was incubated at a final concentration of 0.5 µM with 10 µM QF-peptide substrate in 100 µl of 150 mM NaCl, 10 mM CaCl2 , 50 mM HEPES, pH 7.5, in the presence of HALT protease inhibitor cocktail (Life Technologies) plus/minus 20 mM EDTA at 37 °C. .. Three different quenched fluorescent peptides (PLG↓L), Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg; ALG↓L, Mca-Ala-Leu-Gly-Leu-Dpa-Ala-Arg; and PLG↓V, Mca-Pro-Leu-Gly-Val-Dpa-Ala-Arg) were tested using a multi-wavelength fluorescence scanner (POLARstar OPTIMA, BMG Lab technologies).

    Purification:

    Article Title: Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella
    Article Snippet: Quenched fluorescence protease activity assays Stock solutions (1.0 mM) of synthetic quenched fluorescence (QF) peptide substrates (GenScript Inc.) were dissolved in DMSO and working stocks (100 μM) were prepared using the molar extinction coefficient of the conjugated quencher, (2,4)-dinitrophenyl, of 6.985 cm–1 mM–1 at 400 nm. .. Purified recombinant FliA(H)-hypervariable region protease was incubated at a final concentration of 0.5 µM with 10 µM QF-peptide substrate in 100 µl of 150 mM NaCl, 10 mM CaCl2 , 50 mM HEPES, pH 7.5, in the presence of HALT protease inhibitor cocktail (Life Technologies) plus/minus 20 mM EDTA at 37 °C. .. Three different quenched fluorescent peptides (PLG↓L), Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg; ALG↓L, Mca-Ala-Leu-Gly-Leu-Dpa-Ala-Arg; and PLG↓V, Mca-Pro-Leu-Gly-Val-Dpa-Ala-Arg) were tested using a multi-wavelength fluorescence scanner (POLARstar OPTIMA, BMG Lab technologies).

    Recombinant:

    Article Title: Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella
    Article Snippet: Quenched fluorescence protease activity assays Stock solutions (1.0 mM) of synthetic quenched fluorescence (QF) peptide substrates (GenScript Inc.) were dissolved in DMSO and working stocks (100 μM) were prepared using the molar extinction coefficient of the conjugated quencher, (2,4)-dinitrophenyl, of 6.985 cm–1 mM–1 at 400 nm. .. Purified recombinant FliA(H)-hypervariable region protease was incubated at a final concentration of 0.5 µM with 10 µM QF-peptide substrate in 100 µl of 150 mM NaCl, 10 mM CaCl2 , 50 mM HEPES, pH 7.5, in the presence of HALT protease inhibitor cocktail (Life Technologies) plus/minus 20 mM EDTA at 37 °C. .. Three different quenched fluorescent peptides (PLG↓L), Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg; ALG↓L, Mca-Ala-Leu-Gly-Leu-Dpa-Ala-Arg; and PLG↓V, Mca-Pro-Leu-Gly-Val-Dpa-Ala-Arg) were tested using a multi-wavelength fluorescence scanner (POLARstar OPTIMA, BMG Lab technologies).

    Incubation:

    Article Title: Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella
    Article Snippet: Quenched fluorescence protease activity assays Stock solutions (1.0 mM) of synthetic quenched fluorescence (QF) peptide substrates (GenScript Inc.) were dissolved in DMSO and working stocks (100 μM) were prepared using the molar extinction coefficient of the conjugated quencher, (2,4)-dinitrophenyl, of 6.985 cm–1 mM–1 at 400 nm. .. Purified recombinant FliA(H)-hypervariable region protease was incubated at a final concentration of 0.5 µM with 10 µM QF-peptide substrate in 100 µl of 150 mM NaCl, 10 mM CaCl2 , 50 mM HEPES, pH 7.5, in the presence of HALT protease inhibitor cocktail (Life Technologies) plus/minus 20 mM EDTA at 37 °C. .. Three different quenched fluorescent peptides (PLG↓L), Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg; ALG↓L, Mca-Ala-Leu-Gly-Leu-Dpa-Ala-Arg; and PLG↓V, Mca-Pro-Leu-Gly-Val-Dpa-Ala-Arg) were tested using a multi-wavelength fluorescence scanner (POLARstar OPTIMA, BMG Lab technologies).

    Protein Array:

    Article Title: Th1/M1 Conversion to Th2/M2 Responses in Models of Inflammation Lacking Cell Death Stimulates Maturation of Monocyte Precursors to Fibroblasts
    Article Snippet: Cells were analyzed on a Cell Lab Quanta SC flow cytometer (Beckman Coulter) using the Quanta Analysis software. .. Protein array After 3 days of I/R, hearts were harvested and protein was isolated using Cell Lysis Buffer (RayBiotech) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). .. Protein (250 μg) was loaded onto standard mouse cytokine antibody array membranes (cytokine array C1, RayBiotech).

    Isolation:

    Article Title: Th1/M1 Conversion to Th2/M2 Responses in Models of Inflammation Lacking Cell Death Stimulates Maturation of Monocyte Precursors to Fibroblasts
    Article Snippet: Cells were analyzed on a Cell Lab Quanta SC flow cytometer (Beckman Coulter) using the Quanta Analysis software. .. Protein array After 3 days of I/R, hearts were harvested and protein was isolated using Cell Lysis Buffer (RayBiotech) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). .. Protein (250 μg) was loaded onto standard mouse cytokine antibody array membranes (cytokine array C1, RayBiotech).

    Lysis:

    Article Title: Th1/M1 Conversion to Th2/M2 Responses in Models of Inflammation Lacking Cell Death Stimulates Maturation of Monocyte Precursors to Fibroblasts
    Article Snippet: Cells were analyzed on a Cell Lab Quanta SC flow cytometer (Beckman Coulter) using the Quanta Analysis software. .. Protein array After 3 days of I/R, hearts were harvested and protein was isolated using Cell Lysis Buffer (RayBiotech) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). .. Protein (250 μg) was loaded onto standard mouse cytokine antibody array membranes (cytokine array C1, RayBiotech).

    Article Title: Vasopressin type 1A receptor deletion enhances cardiac contractility, β-adrenergic receptor sensitivity and acute cardiac injury-induced dysfunction
    Article Snippet: A Tecan M1000 multimode plate reader was used to read the absorbance of the samples at 405 nm and Prism 5.0 software (GraphPad Software) was used to calculate the concentration of cAMP in the samples via interpolation from the linear regression of the standard curve. .. Left ventricular samples were homogenized in lysis buffer containing 20 mM Tris (pH 7.4), 137 mM NaCl, 10 % glycerol, 1 mM EDTA, 1 % NP-40, 10 mM NaF (Fisher Scientific), 1× HALT protease inhibitor cocktail (Thermo Scientific) and phosphatase inhibitor cocktail set IV (Calbiochem). .. Lysates were run on SDS/15 % PAGE gels and transferred to Immobilon-PSQ polyvinylidene fluoride 0.2 mm pore size membranes (Millipore), which were then incubated with anti-Phospho-Ser-16 phospholamban (PLB) rabbit pAb (1:5000, Badrilla), anti-PLB mouse mAb (1:1000, Badrilla), anti-Phospho-Ser-23/24 cTnI rabbit pAb (1:1000, Cell Signaling Technology) or anti-cTnI rabbit pAb (1:1000, Cell Signaling Technology).

    Western Blot:

    Article Title: A System for Creating Stable Cell Lines that Express a Gene of Interest from a Bidirectional and Regulatable Herpes Simplex Virus Type 1 Promoter
    Article Snippet: .. Western immunoblotCells were removed from plates with trypsin, washed with PBS and proteins extracted using M-PER (Thermo Scientific) supplemented with Halt Protease inhibitor cocktail (Thermo Scientific). .. After a 30-minute incubation on ice, samples were centrifuged (14,000 rpm/30 min/4°C), supernatants collected and protein concentrations determined by Pierce BCA Protein Assay (Thermo Scientific).

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    Thermo Fisher halt protease inhibitor cocktail
    Peptidase activity assays of purified recombinant FliA(H)-hypervariable region and flagellar filaments from C. haemolyticum . a 12% SDS-PAGE of purified FliA(H)-hypervariable region (band “i”), electrophoresing close to that predicted for recombinant thioredoxin-tagged flagellin hypervariable region (50.14 kDa). b Amino acid occurrence heat map based on FliA(H)-hypervariable region protease cleaved peptide alignments of 391 cleavage sites identified in a tryptic E. coli K12 library. c IceLogos of FliA(H)-hypervariable region protease cleaved peptide specificity profiles showing percent differences compared to natural amino acid abundance, with significantly over-represented amino acids shown above the x -axis and under-represented residues below the x -axis. Amino acids that have not been identified are depicted in pink . d Fluorometric peptidase assays for FliA(H)-hypervariable region protease against three different peptidic substrates (ALG↓L, PLG↓L, PLG↓V) and + /– <t>EDTA</t> as indicated. e 10% SDS-PAGE of purified flagellar sheared filaments from C. haemolyticum . Band “ii” and “iii” were identified by mass spectrometry as FliA(H) (proteolytic flagellin) and the non-protease-containing structural flagellin (WP_039229459), respectively (Supplementary Fig. 7 ). f Amino acid occurrence heat map and g IceLogo based on cleaved peptide alignments of 269 cleavage sites identified in a tryptic E. coli K12 library using purified flagellar filaments. h Fluorometric peptidase assays for flagellar filaments using the ALG↓L peptide in three assay conditions including EDTA-free <t>HALT</t> protease inhibitors
    Halt Protease Inhibitor Cocktail, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/halt protease inhibitor cocktail/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    halt protease inhibitor cocktail - by Bioz Stars, 2021-03
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    97
    Thermo Fisher 1x ethylene diamine triacetic acid edta
    Lentiviral vector is released from LDLR3-ELP after internalization by cells A549 were starved for 24 hours then were treated in serum free media with the indicated treatments. Cells were treated for 24 hours with the lentiviral vector containing nanoparticles that consisted of 1 µM of biotinylated LDLR3-ELP and 1 µM KGF-ELP. At the indicated time points, cells were put on ice for five minutes followed by three ice cold washed in PBS. The cells were lysed with radio immune protection assay (RIPA) buffer containing 2X halt protease and phosphatase inhibitors cocktail with <t>1X</t> ethylene diamine <t>triacetic</t> acid <t>(EDTA).</t> The lysates were immunoprecipitated using streptavidin conjugated magnetic beads (dynabeads). Then a magnetic bar was used to separate the beads from the supernatant. Following a few washes, the immunoprecipitated complexes (dynabeads) and the supernatant were analyzed for the presence of virus using western blots. Anti VSV-G antibody was used to detect the lentiviral vector.
    1x Ethylene Diamine Triacetic Acid Edta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher 1x halt protease
    Analysis of protein expression. Cell-seeded scaffolds were randomly collected, trypsinized, lysed with IP lysis buffer—1X Halt protease and phosphatase inhibitor cocktail and cell lysates were subjected to SDS-PAGE followed by western blotting. (a) In separate experiments, the membranes were incubated with primary antibodies against ACAN, COL1A1, COL2A2, COL9A1, SOX-9, p-SOX-9 (i.e. p-S181-SOX-9). β-Actin was used as the loading control. (b) All protein bands were quantified by ImageJ ™  software and relative protein expression levels were computed with respect to β-actin.
    1x Halt Protease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Peptidase activity assays of purified recombinant FliA(H)-hypervariable region and flagellar filaments from C. haemolyticum . a 12% SDS-PAGE of purified FliA(H)-hypervariable region (band “i”), electrophoresing close to that predicted for recombinant thioredoxin-tagged flagellin hypervariable region (50.14 kDa). b Amino acid occurrence heat map based on FliA(H)-hypervariable region protease cleaved peptide alignments of 391 cleavage sites identified in a tryptic E. coli K12 library. c IceLogos of FliA(H)-hypervariable region protease cleaved peptide specificity profiles showing percent differences compared to natural amino acid abundance, with significantly over-represented amino acids shown above the x -axis and under-represented residues below the x -axis. Amino acids that have not been identified are depicted in pink . d Fluorometric peptidase assays for FliA(H)-hypervariable region protease against three different peptidic substrates (ALG↓L, PLG↓L, PLG↓V) and + /– EDTA as indicated. e 10% SDS-PAGE of purified flagellar sheared filaments from C. haemolyticum . Band “ii” and “iii” were identified by mass spectrometry as FliA(H) (proteolytic flagellin) and the non-protease-containing structural flagellin (WP_039229459), respectively (Supplementary Fig. 7 ). f Amino acid occurrence heat map and g IceLogo based on cleaved peptide alignments of 269 cleavage sites identified in a tryptic E. coli K12 library using purified flagellar filaments. h Fluorometric peptidase assays for flagellar filaments using the ALG↓L peptide in three assay conditions including EDTA-free HALT protease inhibitors

    Journal: Nature Communications

    Article Title: Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella

    doi: 10.1038/s41467-017-00599-0

    Figure Lengend Snippet: Peptidase activity assays of purified recombinant FliA(H)-hypervariable region and flagellar filaments from C. haemolyticum . a 12% SDS-PAGE of purified FliA(H)-hypervariable region (band “i”), electrophoresing close to that predicted for recombinant thioredoxin-tagged flagellin hypervariable region (50.14 kDa). b Amino acid occurrence heat map based on FliA(H)-hypervariable region protease cleaved peptide alignments of 391 cleavage sites identified in a tryptic E. coli K12 library. c IceLogos of FliA(H)-hypervariable region protease cleaved peptide specificity profiles showing percent differences compared to natural amino acid abundance, with significantly over-represented amino acids shown above the x -axis and under-represented residues below the x -axis. Amino acids that have not been identified are depicted in pink . d Fluorometric peptidase assays for FliA(H)-hypervariable region protease against three different peptidic substrates (ALG↓L, PLG↓L, PLG↓V) and + /– EDTA as indicated. e 10% SDS-PAGE of purified flagellar sheared filaments from C. haemolyticum . Band “ii” and “iii” were identified by mass spectrometry as FliA(H) (proteolytic flagellin) and the non-protease-containing structural flagellin (WP_039229459), respectively (Supplementary Fig. 7 ). f Amino acid occurrence heat map and g IceLogo based on cleaved peptide alignments of 269 cleavage sites identified in a tryptic E. coli K12 library using purified flagellar filaments. h Fluorometric peptidase assays for flagellar filaments using the ALG↓L peptide in three assay conditions including EDTA-free HALT protease inhibitors

    Article Snippet: Purified recombinant FliA(H)-hypervariable region protease was incubated at a final concentration of 0.5 µM with 10 µM QF-peptide substrate in 100 µl of 150 mM NaCl, 10 mM CaCl2 , 50 mM HEPES, pH 7.5, in the presence of HALT protease inhibitor cocktail (Life Technologies) plus/minus 20 mM EDTA at 37 °C.

    Techniques: Activity Assay, Purification, Recombinant, SDS Page, Mass Spectrometry

    Lentiviral vector is released from LDLR3-ELP after internalization by cells A549 were starved for 24 hours then were treated in serum free media with the indicated treatments. Cells were treated for 24 hours with the lentiviral vector containing nanoparticles that consisted of 1 µM of biotinylated LDLR3-ELP and 1 µM KGF-ELP. At the indicated time points, cells were put on ice for five minutes followed by three ice cold washed in PBS. The cells were lysed with radio immune protection assay (RIPA) buffer containing 2X halt protease and phosphatase inhibitors cocktail with 1X ethylene diamine triacetic acid (EDTA). The lysates were immunoprecipitated using streptavidin conjugated magnetic beads (dynabeads). Then a magnetic bar was used to separate the beads from the supernatant. Following a few washes, the immunoprecipitated complexes (dynabeads) and the supernatant were analyzed for the presence of virus using western blots. Anti VSV-G antibody was used to detect the lentiviral vector.

    Journal: Gene therapy

    Article Title: Recombinant elastin based nanoparticles for targeted gene therapy

    doi: 10.1038/gt.2017.54

    Figure Lengend Snippet: Lentiviral vector is released from LDLR3-ELP after internalization by cells A549 were starved for 24 hours then were treated in serum free media with the indicated treatments. Cells were treated for 24 hours with the lentiviral vector containing nanoparticles that consisted of 1 µM of biotinylated LDLR3-ELP and 1 µM KGF-ELP. At the indicated time points, cells were put on ice for five minutes followed by three ice cold washed in PBS. The cells were lysed with radio immune protection assay (RIPA) buffer containing 2X halt protease and phosphatase inhibitors cocktail with 1X ethylene diamine triacetic acid (EDTA). The lysates were immunoprecipitated using streptavidin conjugated magnetic beads (dynabeads). Then a magnetic bar was used to separate the beads from the supernatant. Following a few washes, the immunoprecipitated complexes (dynabeads) and the supernatant were analyzed for the presence of virus using western blots. Anti VSV-G antibody was used to detect the lentiviral vector.

    Article Snippet: The cells were then lysed with radio immune protection assay (RIPA) buffer containing 2X halt protease and phosphatase inhibitors cocktail with 1X ethylene diamine triacetic acid (EDTA) (Thermo scientific cat #78440).

    Techniques: Plasmid Preparation, Immunoprecipitation, Magnetic Beads, Western Blot

    N-Cadherin from POMGNT1-deficient cells shows enhanced homotypic interactions in vitro . (A) Semi-quantitative analysis of the mean size of dynabead aggregates coated with the recombinant extracellular domain of recombinant N-Cdh purified from WT (gray line) and ΔPOMGnT1 cells (black line). Protein-coated dynabeads were imaged following incubation in the presence of either CaCl 2 (solid lines) or EDTA (broken lines) at the indicated time intervals. Mean aggregate size is plotted against time. Assays were performed at quadruplicate from two independent experiments. Data are represented as means ± SD. Asterisks denote statistical significance in comparison to WT cells: * p ≤ 0.05, ** p ≤ 0.01. (B) Western Blot analysis evaluating levels of protein from WT and ΔPOMGnT1 (Δ) bound to dynabeads. Dynabead-bound recombinant N-Cdh was eluted in SDS sample buffer (beads), resolved on a 10% SDS PAA gel. For detection on Western blot an anti-5xHis antibody was used. In addition, protein before binding to beads (input) and unbound fractions (void) are shown.

    Journal: bioRxiv

    Article Title: Glycosyltransferase POMGNT1 deficiency affects N-cadherin-mediated cell-cell adhesion

    doi: 10.1101/2020.09.09.289306

    Figure Lengend Snippet: N-Cadherin from POMGNT1-deficient cells shows enhanced homotypic interactions in vitro . (A) Semi-quantitative analysis of the mean size of dynabead aggregates coated with the recombinant extracellular domain of recombinant N-Cdh purified from WT (gray line) and ΔPOMGnT1 cells (black line). Protein-coated dynabeads were imaged following incubation in the presence of either CaCl 2 (solid lines) or EDTA (broken lines) at the indicated time intervals. Mean aggregate size is plotted against time. Assays were performed at quadruplicate from two independent experiments. Data are represented as means ± SD. Asterisks denote statistical significance in comparison to WT cells: * p ≤ 0.05, ** p ≤ 0.01. (B) Western Blot analysis evaluating levels of protein from WT and ΔPOMGnT1 (Δ) bound to dynabeads. Dynabead-bound recombinant N-Cdh was eluted in SDS sample buffer (beads), resolved on a 10% SDS PAA gel. For detection on Western blot an anti-5xHis antibody was used. In addition, protein before binding to beads (input) and unbound fractions (void) are shown.

    Article Snippet: Whole cell lysate and crude membrane preparation Cells at 90% confluency (~5.0 x 106 cells) were washed with ice-cold PBS and lysed with 500 μl of lysis buffer (10 mM Tris-HCl pH 7.6, 1% (w/v) SDS, 1 mM EDTA, supplemented with Halt™ protease and phosphatase inhibitor cocktail without EDTA (ThermoFisher Scientific)).

    Techniques: In Vitro, Recombinant, Purification, Incubation, Western Blot, Binding Assay

    Analysis of protein expression. Cell-seeded scaffolds were randomly collected, trypsinized, lysed with IP lysis buffer—1X Halt protease and phosphatase inhibitor cocktail and cell lysates were subjected to SDS-PAGE followed by western blotting. (a) In separate experiments, the membranes were incubated with primary antibodies against ACAN, COL1A1, COL2A2, COL9A1, SOX-9, p-SOX-9 (i.e. p-S181-SOX-9). β-Actin was used as the loading control. (b) All protein bands were quantified by ImageJ ™  software and relative protein expression levels were computed with respect to β-actin.

    Journal: Journal of Tissue Engineering

    Article Title: Growth factor and ultrasound-assisted bioreactor synergism for human mesenchymal stem cell chondrogenesis

    doi: 10.1177/2041731414566529

    Figure Lengend Snippet: Analysis of protein expression. Cell-seeded scaffolds were randomly collected, trypsinized, lysed with IP lysis buffer—1X Halt protease and phosphatase inhibitor cocktail and cell lysates were subjected to SDS-PAGE followed by western blotting. (a) In separate experiments, the membranes were incubated with primary antibodies against ACAN, COL1A1, COL2A2, COL9A1, SOX-9, p-SOX-9 (i.e. p-S181-SOX-9). β-Actin was used as the loading control. (b) All protein bands were quantified by ImageJ ™ software and relative protein expression levels were computed with respect to β-actin.

    Article Snippet: Protein isolation and western blotting analysis At the end of 14 days of culture, cells were released from scaffolds with 1X Trypsin followed by pelleting cells at 1000×g and finally lysing the pellet with Pierce IP lysis buffer supplemented with 1X Halt protease and phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL, USA).

    Techniques: Expressing, Lysis, SDS Page, Western Blot, Incubation, Software