ez link phosphine peg3 biotin  (Millipore)


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    Structured Review

    Millipore ez link phosphine peg3 biotin
    Ez Link Phosphine Peg3 Biotin, supplied by Millipore, used in various techniques. Bioz Stars score: 83/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ez link phosphine peg3 biotin/product/Millipore
    Average 83 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ez link phosphine peg3 biotin - by Bioz Stars, 2020-01
    83/100 stars

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    Related Articles

    Sample Prep:

    Article Title: Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics
    Article Snippet: Paragraph title: Sample preparation and MS-based proteomic analysis ... PL3 -labeled samples were instead conjugated to EZ-Link™ Phosphine-PEG3 -Biotin (Sigma Aldrich) via Staudinger ligation (0.2 mM per 1 ml sample, 2 mg protein, 4 h at 37°C, then 20 h at 25°C).

    In Situ:

    Article Title: Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics
    Article Snippet: Sample preparation and MS-based proteomic analysis ( 2h in situ protocol ): A stationary phase culture of S. aureus USA300 TnPdxS (Nebraska transposon mutant library) grown in B-medium was exchanged into chemically-defined (PL-free) medium (CDM) containing 5 µg/ml Erythromycin and adjusted to OD600 = 40. .. PL3 -labeled samples were instead conjugated to EZ-Link™ Phosphine-PEG3 -Biotin (Sigma Aldrich) via Staudinger ligation (0.2 mM per 1 ml sample, 2 mg protein, 4 h at 37°C, then 20 h at 25°C).

    Ligation:

    Article Title: Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics
    Article Snippet: .. PL3 -labeled samples were instead conjugated to EZ-Link™ Phosphine-PEG3 -Biotin (Sigma Aldrich) via Staudinger ligation (0.2 mM per 1 ml sample, 2 mg protein, 4 h at 37°C, then 20 h at 25°C). .. Upon avidin-bead enrichment, samples were reduced with 5 mM TCEP (1 h at 37°C), alkylated using 10 mM IAA (30 min at 25°C) and quenched with 10 mM DTT (30 min at 25°C) on-bead.

    Mutagenesis:

    Article Title: Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics
    Article Snippet: Sample preparation and MS-based proteomic analysis ( 2h in situ protocol ): A stationary phase culture of S. aureus USA300 TnPdxS (Nebraska transposon mutant library) grown in B-medium was exchanged into chemically-defined (PL-free) medium (CDM) containing 5 µg/ml Erythromycin and adjusted to OD600 = 40. .. PL3 -labeled samples were instead conjugated to EZ-Link™ Phosphine-PEG3 -Biotin (Sigma Aldrich) via Staudinger ligation (0.2 mM per 1 ml sample, 2 mg protein, 4 h at 37°C, then 20 h at 25°C).

    Avidin-Biotin Assay:

    Article Title: Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics
    Article Snippet: PL3 -labeled samples were instead conjugated to EZ-Link™ Phosphine-PEG3 -Biotin (Sigma Aldrich) via Staudinger ligation (0.2 mM per 1 ml sample, 2 mg protein, 4 h at 37°C, then 20 h at 25°C). .. Upon avidin-bead enrichment, samples were reduced with 5 mM TCEP (1 h at 37°C), alkylated using 10 mM IAA (30 min at 25°C) and quenched with 10 mM DTT (30 min at 25°C) on-bead.

    Labeling:

    Article Title: Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics
    Article Snippet: .. PL3 -labeled samples were instead conjugated to EZ-Link™ Phosphine-PEG3 -Biotin (Sigma Aldrich) via Staudinger ligation (0.2 mM per 1 ml sample, 2 mg protein, 4 h at 37°C, then 20 h at 25°C). .. Upon avidin-bead enrichment, samples were reduced with 5 mM TCEP (1 h at 37°C), alkylated using 10 mM IAA (30 min at 25°C) and quenched with 10 mM DTT (30 min at 25°C) on-bead.

    Incubation:

    Article Title: Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics
    Article Snippet: For each sample, 1 ml of this suspension (≈ 1 mg protein) was incubated with 10 µl probe or DMSO (100× stock in DMSO) for 2 h at 37°C and bacteria were washed with PBS prior to cell lysis. ( ): Overnight cultures of S. aureus USA300 TnPdxS grown in B-medium were washed in CDM-Erm and used to inoculate cultures (to OD600 = 0.1) in CDM-Erm (100 ml) supplemented with 25 µM probe (25 µl of 100 mM stock in DMSO) or 0.5 µM PL (25 µl of 2 mM stock in DMSO) in baffled flasks. .. PL3 -labeled samples were instead conjugated to EZ-Link™ Phosphine-PEG3 -Biotin (Sigma Aldrich) via Staudinger ligation (0.2 mM per 1 ml sample, 2 mg protein, 4 h at 37°C, then 20 h at 25°C).

    Mass Spectrometry:

    Article Title: Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics
    Article Snippet: Paragraph title: Sample preparation and MS-based proteomic analysis ... PL3 -labeled samples were instead conjugated to EZ-Link™ Phosphine-PEG3 -Biotin (Sigma Aldrich) via Staudinger ligation (0.2 mM per 1 ml sample, 2 mg protein, 4 h at 37°C, then 20 h at 25°C).

    Lysis:

    Article Title: Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics
    Article Snippet: Bacteria were grown to stationary phase (30 h at 37°C), harvested, washed with PBS and adjusted to 1 ml OD600 = 60 (≈ 2 mg protein) prior to cell lysis. .. PL3 -labeled samples were instead conjugated to EZ-Link™ Phosphine-PEG3 -Biotin (Sigma Aldrich) via Staudinger ligation (0.2 mM per 1 ml sample, 2 mg protein, 4 h at 37°C, then 20 h at 25°C).

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    Millipore aml5 peg3
    Molecular-level activation of Met signalling pathway by dimeric macrocycles. ( a ) Met activation by the dimeric macrocycles initiates pathways analogous to hHGF-activated pathways. Each dimeric macrocycle was chemically synthesized by conjugating the C-terminal Cys residues using a bis-maleimide cross-linker ( Supplementary Table 2 ) and forms a 1:2 stoichiometric complex with Met. ( b – d ) Met phosphorylation level as a function of hHGF concentration (red circles) as a positive control; ( b ) <t>aML5-C6</t> (blue triangles), <t>aML5-PEG3</t> (blue rhombuses) and aML5-PEG11 (blue squares); ( c ) aMD4-C6 (orange triangles), aMD4-PEG3 (orange rhombuses), aMD4-PEG11 (orange squares) and aMsD4-C6 (grey triangles) as a negative control; and ( d ) aMD5-C6 (green triangles), aMD5-PEG3 (green rhombuses) and aMD5-PEG11 (green squares). The phosphorylation level of Met in each experiment was quantified by a cell-based phospho-Met ELISA 10 min after cell stimulation. s.d.was calculated from the results of triplicate experiments. ( e ) Analysis of Met dimerization by cross-linking of Met dimer on live cells. EHMES-1 cells were treated with dimeric macrocycles for 60 min at 4 °C. After washing, the cell surface proteins were cross-linked by BS3 cross-linker for 60 min at 4 °C. Cell lysates were subjected to immunoprecipitation and western blotting using an anti-Met antibody. Arrowheads and arrows indicate Met dimer and monomer corresponding to cross-linked αβ subunits, respectively. Asterisks indicate Met monomer corresponding to β-subunit. Each bar in the graph indicates a relative value of the cross-linked Met dimer generated by the band intensity. ( f ) Phosphorylation of Met and downstream signalling proteins. Starved EHMES-1 cells were treated with hHGF (2 nM) or dimeric macrocycles (each at 100 nM) for 10 min and phosphorylation of the respective proteins was analysed by western blotting using their specific antibodies. ( g ) Phosphorylation of various RTKs stimulated by dimeric macrocycles. Lysates of EHMES-1 cells stimulated by 100 nM of dimeric macrocycles or 2 nM of hHGF for 10 min were analysed by a phospho-RTK array of 49 representative human RTKs. Both hHGF and dimeric macrocycles specifically phosphorylated Met (delineated by red boxes).
    Aml5 Peg3, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/aml5 peg3/product/Millipore
    Average 78 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    aml5 peg3 - by Bioz Stars, 2020-01
    78/100 stars
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    Molecular-level activation of Met signalling pathway by dimeric macrocycles. ( a ) Met activation by the dimeric macrocycles initiates pathways analogous to hHGF-activated pathways. Each dimeric macrocycle was chemically synthesized by conjugating the C-terminal Cys residues using a bis-maleimide cross-linker ( Supplementary Table 2 ) and forms a 1:2 stoichiometric complex with Met. ( b – d ) Met phosphorylation level as a function of hHGF concentration (red circles) as a positive control; ( b ) aML5-C6 (blue triangles), aML5-PEG3 (blue rhombuses) and aML5-PEG11 (blue squares); ( c ) aMD4-C6 (orange triangles), aMD4-PEG3 (orange rhombuses), aMD4-PEG11 (orange squares) and aMsD4-C6 (grey triangles) as a negative control; and ( d ) aMD5-C6 (green triangles), aMD5-PEG3 (green rhombuses) and aMD5-PEG11 (green squares). The phosphorylation level of Met in each experiment was quantified by a cell-based phospho-Met ELISA 10 min after cell stimulation. s.d.was calculated from the results of triplicate experiments. ( e ) Analysis of Met dimerization by cross-linking of Met dimer on live cells. EHMES-1 cells were treated with dimeric macrocycles for 60 min at 4 °C. After washing, the cell surface proteins were cross-linked by BS3 cross-linker for 60 min at 4 °C. Cell lysates were subjected to immunoprecipitation and western blotting using an anti-Met antibody. Arrowheads and arrows indicate Met dimer and monomer corresponding to cross-linked αβ subunits, respectively. Asterisks indicate Met monomer corresponding to β-subunit. Each bar in the graph indicates a relative value of the cross-linked Met dimer generated by the band intensity. ( f ) Phosphorylation of Met and downstream signalling proteins. Starved EHMES-1 cells were treated with hHGF (2 nM) or dimeric macrocycles (each at 100 nM) for 10 min and phosphorylation of the respective proteins was analysed by western blotting using their specific antibodies. ( g ) Phosphorylation of various RTKs stimulated by dimeric macrocycles. Lysates of EHMES-1 cells stimulated by 100 nM of dimeric macrocycles or 2 nM of hHGF for 10 min were analysed by a phospho-RTK array of 49 representative human RTKs. Both hHGF and dimeric macrocycles specifically phosphorylated Met (delineated by red boxes).

    Journal: Nature Communications

    Article Title: Artificial human Met agonists based on macrocycle scaffolds

    doi: 10.1038/ncomms7373

    Figure Lengend Snippet: Molecular-level activation of Met signalling pathway by dimeric macrocycles. ( a ) Met activation by the dimeric macrocycles initiates pathways analogous to hHGF-activated pathways. Each dimeric macrocycle was chemically synthesized by conjugating the C-terminal Cys residues using a bis-maleimide cross-linker ( Supplementary Table 2 ) and forms a 1:2 stoichiometric complex with Met. ( b – d ) Met phosphorylation level as a function of hHGF concentration (red circles) as a positive control; ( b ) aML5-C6 (blue triangles), aML5-PEG3 (blue rhombuses) and aML5-PEG11 (blue squares); ( c ) aMD4-C6 (orange triangles), aMD4-PEG3 (orange rhombuses), aMD4-PEG11 (orange squares) and aMsD4-C6 (grey triangles) as a negative control; and ( d ) aMD5-C6 (green triangles), aMD5-PEG3 (green rhombuses) and aMD5-PEG11 (green squares). The phosphorylation level of Met in each experiment was quantified by a cell-based phospho-Met ELISA 10 min after cell stimulation. s.d.was calculated from the results of triplicate experiments. ( e ) Analysis of Met dimerization by cross-linking of Met dimer on live cells. EHMES-1 cells were treated with dimeric macrocycles for 60 min at 4 °C. After washing, the cell surface proteins were cross-linked by BS3 cross-linker for 60 min at 4 °C. Cell lysates were subjected to immunoprecipitation and western blotting using an anti-Met antibody. Arrowheads and arrows indicate Met dimer and monomer corresponding to cross-linked αβ subunits, respectively. Asterisks indicate Met monomer corresponding to β-subunit. Each bar in the graph indicates a relative value of the cross-linked Met dimer generated by the band intensity. ( f ) Phosphorylation of Met and downstream signalling proteins. Starved EHMES-1 cells were treated with hHGF (2 nM) or dimeric macrocycles (each at 100 nM) for 10 min and phosphorylation of the respective proteins was analysed by western blotting using their specific antibodies. ( g ) Phosphorylation of various RTKs stimulated by dimeric macrocycles. Lysates of EHMES-1 cells stimulated by 100 nM of dimeric macrocycles or 2 nM of hHGF for 10 min were analysed by a phospho-RTK array of 49 representative human RTKs. Both hHGF and dimeric macrocycles specifically phosphorylated Met (delineated by red boxes).

    Article Snippet: The cells were left untreated or stimulated with hHGF or aML5-PEG3 in the presence or absence of the indicated concentrations of the specific Met kinase inhibitor, SU11274 (Calbiochem).

    Techniques: Activation Assay, Synthesized, Concentration Assay, Positive Control, Negative Control, Enzyme-linked Immunosorbent Assay, Cell Stimulation, Immunoprecipitation, Western Blot, Generated

    Cellular migration and wound healing promoted by dimeric macrocycles. ( a ) Captured images for migration of normal human RPTEC stimulated by hHGF (0.26 nM) or dimer macrocycles (20 nM) over 30 h. Cells were stained by calcein-AM. Scale bar, 200 μm. ( b ) Dose-dependent titration of cell migration stimulated by 0.4 pM–30 nM of hHGF (red), 40 pM–3,000 nM of aML5-PEG3 (blue), aMD4-PEG11 (orange), aMsD4-C6 (grey) as a negative control, or aMD5-PEG11 (green). Migrated cells were stained with calcein-AM and quantified by fluorescence intensity. s.d. was calculated from the results of experiments in triplicate. ( c ) Captured images of wound healing in NHEK promoted by various stimulants (also see Supplementary Movie 1 ). Wound-closure events in the presence or the absence of 0.25 nM hHGF or 100 nM dimeric macrocycles were monitored by a real-time cultured cell monitoring system. The images were taken at 4 and 50 h. Red broken lines indicate boundaries between cells in the monolayer and the scratched areas uncovered by cells. Scale bar, 500 μm. ( d ) Quantification of relative wound-closure areas. Error bars denote s.e.m. ( n =3). * P

    Journal: Nature Communications

    Article Title: Artificial human Met agonists based on macrocycle scaffolds

    doi: 10.1038/ncomms7373

    Figure Lengend Snippet: Cellular migration and wound healing promoted by dimeric macrocycles. ( a ) Captured images for migration of normal human RPTEC stimulated by hHGF (0.26 nM) or dimer macrocycles (20 nM) over 30 h. Cells were stained by calcein-AM. Scale bar, 200 μm. ( b ) Dose-dependent titration of cell migration stimulated by 0.4 pM–30 nM of hHGF (red), 40 pM–3,000 nM of aML5-PEG3 (blue), aMD4-PEG11 (orange), aMsD4-C6 (grey) as a negative control, or aMD5-PEG11 (green). Migrated cells were stained with calcein-AM and quantified by fluorescence intensity. s.d. was calculated from the results of experiments in triplicate. ( c ) Captured images of wound healing in NHEK promoted by various stimulants (also see Supplementary Movie 1 ). Wound-closure events in the presence or the absence of 0.25 nM hHGF or 100 nM dimeric macrocycles were monitored by a real-time cultured cell monitoring system. The images were taken at 4 and 50 h. Red broken lines indicate boundaries between cells in the monolayer and the scratched areas uncovered by cells. Scale bar, 500 μm. ( d ) Quantification of relative wound-closure areas. Error bars denote s.e.m. ( n =3). * P

    Article Snippet: The cells were left untreated or stimulated with hHGF or aML5-PEG3 in the presence or absence of the indicated concentrations of the specific Met kinase inhibitor, SU11274 (Calbiochem).

    Techniques: Migration, Staining, Titration, Negative Control, Fluorescence, Cell Culture

    Cellular proliferation promoted by the dimeric macrocycles in normal human cells. ( a–c ) Dose-dependent titration of proliferation induction in normal human epidermal keratinocytes (NHEK) against concentration of hHGF (red) as a positive control, ( a ) aML5-PEG3 (blue), ( b ) aMD4-PEG11 (orange) and aMsD4-C6 (grey) as a negative control, or ( c ) aMD5-PEG11 (green). NHEK were cultured for 6 days with or without hHGF (0.4 pM–30 nM) or dimeric macrocycles (40 pM–3,000 nM). Cells were stained by calcein-AM and quantified by fluorescence intensity. s.d. was calculated from the results of experiments in triplicate. ( d ) Time course of proliferation induction in NHEK. NHEK were cultured with or without 1.3 nM hHGF or 50 nM dimeric macrocycles. After 2, 4 and 6 days, cell numbers were counted by means of an automated cell counter (mean±s.d., n =3). * P

    Journal: Nature Communications

    Article Title: Artificial human Met agonists based on macrocycle scaffolds

    doi: 10.1038/ncomms7373

    Figure Lengend Snippet: Cellular proliferation promoted by the dimeric macrocycles in normal human cells. ( a–c ) Dose-dependent titration of proliferation induction in normal human epidermal keratinocytes (NHEK) against concentration of hHGF (red) as a positive control, ( a ) aML5-PEG3 (blue), ( b ) aMD4-PEG11 (orange) and aMsD4-C6 (grey) as a negative control, or ( c ) aMD5-PEG11 (green). NHEK were cultured for 6 days with or without hHGF (0.4 pM–30 nM) or dimeric macrocycles (40 pM–3,000 nM). Cells were stained by calcein-AM and quantified by fluorescence intensity. s.d. was calculated from the results of experiments in triplicate. ( d ) Time course of proliferation induction in NHEK. NHEK were cultured with or without 1.3 nM hHGF or 50 nM dimeric macrocycles. After 2, 4 and 6 days, cell numbers were counted by means of an automated cell counter (mean±s.d., n =3). * P

    Article Snippet: The cells were left untreated or stimulated with hHGF or aML5-PEG3 in the presence or absence of the indicated concentrations of the specific Met kinase inhibitor, SU11274 (Calbiochem).

    Techniques: Titration, Concentration Assay, Positive Control, Negative Control, Cell Culture, Staining, Fluorescence