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  • 93
    Beijing CWBio pbst
    Pbst, supplied by Beijing CWBio, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/Beijing CWBio
    Average 93 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    93/100 stars
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    92
    Jackson Immuno pbst
    Pbst, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 92/100, based on 467 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/Jackson Immuno
    Average 92 stars, based on 467 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    92/100 stars
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    96
    Thermo Fisher pbst
    Pbst, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1467 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/Thermo Fisher
    Average 96 stars, based on 1467 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    96/100 stars
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    94
    Cell Signaling Technology Inc pbst
    Pbst, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/Cell Signaling Technology Inc
    Average 94 stars, based on 212 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    94/100 stars
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    91
    Cellgro pbst
    Pbst, supplied by Cellgro, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/Cellgro
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    91/100 stars
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    94
    Millipore pbst
    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
    Pbst, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 882 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/Millipore
    Average 94 stars, based on 882 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    94/100 stars
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    92
    The Jackson Laboratory pbst
    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
    Pbst, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 92/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/The Jackson Laboratory
    Average 92 stars, based on 108 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    92/100 stars
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    91
    Trion Research pbst
    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
    Pbst, supplied by Trion Research, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/Trion Research
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    91/100 stars
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    92
    Agilent technologies pbst
    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
    Pbst, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/Agilent technologies
    Average 92 stars, based on 107 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    92/100 stars
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    93
    Fisher Scientific pbst
    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
    Pbst, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 93/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/Fisher Scientific
    Average 93 stars, based on 54 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    93/100 stars
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    92
    FUJIFILM pbst
    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
    Pbst, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/FUJIFILM
    Average 92 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    92/100 stars
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    91
    G Biosciences pbst
    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
    Pbst, supplied by G Biosciences, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/G Biosciences
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    91/100 stars
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    91
    US Biological Life Sciences pbst
    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
    Pbst, supplied by US Biological Life Sciences, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/US Biological Life Sciences
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    91/100 stars
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    94
    Developmental Studies Hybridoma Bank pbst
    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
    Pbst, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/Developmental Studies Hybridoma Bank
    Average 94 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    94/100 stars
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    94
    SouthernBiotech pbst
    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
    Pbst, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 94/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/SouthernBiotech
    Average 94 stars, based on 105 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    94/100 stars
      Buy from Supplier

    92
    Bio-Rad pbst
    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
    Pbst, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/Bio-Rad
    Average 92 stars, based on 138 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    92/100 stars
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    pbst  (Abcam)
    92
    Abcam pbst
    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
    Pbst, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 239 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/Abcam
    Average 92 stars, based on 239 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    92/100 stars
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    91
    AvesLabs pbst
    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
    Pbst, supplied by AvesLabs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbst/product/AvesLabs
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbst - by Bioz Stars, 2020-05
    91/100 stars
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    92
    Becton Dickinson pbst
    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
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    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
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    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
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    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
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    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
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    pbst  (Roche)
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    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
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    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and <t>FLAG-ubiquitin</t> or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with <t>PBST</t> before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c
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    High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and FLAG-ubiquitin or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with PBST before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c

    Journal: Cell Death & Disease

    Article Title: High throughput screening for inhibitors of the HECT ubiquitin E3 ligase ITCH identifies antidepressant drugs as regulators of autophagy

    doi: 10.1038/cddis.2014.113

    Figure Lengend Snippet: High throughput screening (HTS) for ITCH inhibitors. ( a ) Schematic representation of the layout of the ELISA assay used for the HTS. Recombinant GST-ITCH or ubiquitin ligase dead mutant GST-ITCH C830A were immobilized to glutathione-coated ELISA plates and incubated with either complete ubiquitylation reaction mixtures containing E1, E2 (UbcH7) and FLAG-ubiquitin or partial mixtures as indicated in ( b ). Reactions were performed for 1 h at RT and stopped by washing with PBST before development with HRP-conjugated anti-FLAG antibody for 1 h at RT. After final washes, the wells were incubated with TMB substrate for 15 min at RT, and then stopped with acid and OD450 nm measurements were made with a plate reader. ( b ) Different combinations of the ubiquitin reaction components were used to evaluate the specificity of the ubiquitylation reaction (E3, GST-ITCH; E3m, E3 ligase dead mutant GST-ITCH C830A). ( c ) Complete reactions were performed as in ( b ) using a range of E3 or E3m concentrations immobilized to the ELISA plate as shown. ( d ) Normalized screening data for 1040 compounds from the NINDS library. Immobilized GST-ITCH was incubated with test compounds (10 μ M) and E1/E2/Ub mix. After incubation at RT for 2 h, plates were developed with HRP-conjugated anti-FLAG and TMB substrate as above. Percentage residual activity (%RA) was calculated using the formula %RA=(OD450 sample—mean OD450 low controls)/(mean high controls—mean OD450 low controls) × 100. Z prime values were calculated with a threshold of > 0.5 set for acceptable data. 70 and 50% thresholds are indicated with lines. ( e ) Confirmation of six positive hits using does response and the ELISA system as in Figure 1c

    Article Snippet: Bound FLAG peptide corresponding to incorporation into the E3 was detected by addition of 20 μ l anti-FLAG M2 monoclonal antibody conjugated to horseradish peroxidise (Sigma) diluted 1/10,000 in PBST and incubated for 1 h at RT, followed by three washes with PBST and addition of 20 μ l 0.1% w/v 3,3′′,5,5′′-TMB (Sigma) in 0.1 M sodium acetate buffer pH 4.5 containing 0.01% v/v hydrogen peroxide for 15 min.

    Techniques: High Throughput Screening Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Mutagenesis, Incubation, Activity Assay