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itc buffer  (Thermo Fisher)


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    Structured Review

    Thermo Fisher itc buffer
    ( a ) Characterisation of BRD4 mutants that disrupt binding to CDK9-cyclin T1. Mutation of residues Gln1350, Leu1354 and Phe1357, highlighted by the FragLite map and AlphaFold3 model disrupt BRD4 binding to CDK9-Cyclin T1. ( b ) Mutation of BRD4 residues Leu1354 and Phe1357, leads to a loss of BRD4 binding to CDK9-cyclin T2 in a fluorescence polarisation (FP) assay. ( c ) The cyclin T1 Tyr175Ala mutation reduces the Homogenous Time-Resolved Fluorescence (HTRF) signal, whereas the Trp210Ala mutation, previously identified as important for AFF4 interaction and adjacent to Tyr175, shows signals comparable to wild-type. ( d-g ) <t>Isothermal</t> <t>Titration</t> <t>Calorimetry</t> <t>(ITC)</t> plots of CDK9-cyclin T2 complexes with BRD4. Representative titration plots for (d) CDK9-cyclin T2 (e) CDK9-cyclin T2 Tyr174Ala, (f) CDK9-cyclin T2 Phe175Ala (negative control), and (g) CDK9-cyclin T2 Trp206Ala vs the BRD4 P-TEFb Interaction Domain (PID). The top panels show the raw heat signal, and the bottom panels show the integrated heat per injection fitted to a single-site binding model. HTRF experiments were carried out in triplicate and repeated on three separate days. The error bars indicate SD. FP experiments were carried out in triplicate and repeated on three separate days. ITC thermodynamic parameters (K d ) were derived from three independent biological replicates. Derived K d values are compiled in . Related to and Supplementary Figure 11.
    Itc Buffer, 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
    https://www.bioz.com/product/itc+buffer/HEPES/bio_rxiv__64898__2026__04__09__717428-290-14-35
    Average 99 stars, based on 1 article reviews
    itc buffer - by Bioz Stars, 2026-09
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    Images

    1) Product Images from "FragLite mapping to identify the BRD4 recruitment site of P-TEFb"

    Article Title: FragLite mapping to identify the BRD4 recruitment site of P-TEFb

    Journal: bioRxiv

    doi: 10.64898/2026.04.09.717428

    ( a ) Characterisation of BRD4 mutants that disrupt binding to CDK9-cyclin T1. Mutation of residues Gln1350, Leu1354 and Phe1357, highlighted by the FragLite map and AlphaFold3 model disrupt BRD4 binding to CDK9-Cyclin T1. ( b ) Mutation of BRD4 residues Leu1354 and Phe1357, leads to a loss of BRD4 binding to CDK9-cyclin T2 in a fluorescence polarisation (FP) assay. ( c ) The cyclin T1 Tyr175Ala mutation reduces the Homogenous Time-Resolved Fluorescence (HTRF) signal, whereas the Trp210Ala mutation, previously identified as important for AFF4 interaction and adjacent to Tyr175, shows signals comparable to wild-type. ( d-g ) Isothermal Titration Calorimetry (ITC) plots of CDK9-cyclin T2 complexes with BRD4. Representative titration plots for (d) CDK9-cyclin T2 (e) CDK9-cyclin T2 Tyr174Ala, (f) CDK9-cyclin T2 Phe175Ala (negative control), and (g) CDK9-cyclin T2 Trp206Ala vs the BRD4 P-TEFb Interaction Domain (PID). The top panels show the raw heat signal, and the bottom panels show the integrated heat per injection fitted to a single-site binding model. HTRF experiments were carried out in triplicate and repeated on three separate days. The error bars indicate SD. FP experiments were carried out in triplicate and repeated on three separate days. ITC thermodynamic parameters (K d ) were derived from three independent biological replicates. Derived K d values are compiled in . Related to and Supplementary Figure 11.
    Figure Legend Snippet: ( a ) Characterisation of BRD4 mutants that disrupt binding to CDK9-cyclin T1. Mutation of residues Gln1350, Leu1354 and Phe1357, highlighted by the FragLite map and AlphaFold3 model disrupt BRD4 binding to CDK9-Cyclin T1. ( b ) Mutation of BRD4 residues Leu1354 and Phe1357, leads to a loss of BRD4 binding to CDK9-cyclin T2 in a fluorescence polarisation (FP) assay. ( c ) The cyclin T1 Tyr175Ala mutation reduces the Homogenous Time-Resolved Fluorescence (HTRF) signal, whereas the Trp210Ala mutation, previously identified as important for AFF4 interaction and adjacent to Tyr175, shows signals comparable to wild-type. ( d-g ) Isothermal Titration Calorimetry (ITC) plots of CDK9-cyclin T2 complexes with BRD4. Representative titration plots for (d) CDK9-cyclin T2 (e) CDK9-cyclin T2 Tyr174Ala, (f) CDK9-cyclin T2 Phe175Ala (negative control), and (g) CDK9-cyclin T2 Trp206Ala vs the BRD4 P-TEFb Interaction Domain (PID). The top panels show the raw heat signal, and the bottom panels show the integrated heat per injection fitted to a single-site binding model. HTRF experiments were carried out in triplicate and repeated on three separate days. The error bars indicate SD. FP experiments were carried out in triplicate and repeated on three separate days. ITC thermodynamic parameters (K d ) were derived from three independent biological replicates. Derived K d values are compiled in . Related to and Supplementary Figure 11.

    Techniques Used: Binding Assay, Mutagenesis, Fluorescence, FP Assay, Isothermal Titration Calorimetry, Titration, Negative Control, Injection, Derivative Assay

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    Article Snippet: .. The buffer of purified recombinant GFP, GST-nb-GFP, mCherry, and GST-nb-mCherry was exchanged into ITC buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, pH 7.4) using 7K MWCO ZebaTM spin desalting columns (ThermoFisher Scientific). .. ITC measurements were carried out at 25 ◦C using a MicroCal PEAQ-ITC instrument (Malvern Panalytical, Worcestershire, United Kingdom).

    Article Title: A Simplified Strategy for Nanobody Production and Use Based on Functional GST-Nanobody Fusion Proteins
    Article Snippet: .. The buffer of purified recombinant GFP, GST-nb-GFP, mCherry, and GST-nb-mCherry was exchanged into ITC buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, pH 7.4) using 7K MWCO Zeba TM spin desalting columns (ThermoFisher Scientific). .. ITC measurements were carried out at 25 °C using a MicroCal PEAQ-ITC instrument (Malvern Panalytical, Worcestershire, United Kingdom).

    Molecular Weight:

    Article Title: Design, synthesis, biological and computational evaluation of novel S/N-glycerolyl and peptide-conjugated [1,2,4]triazolo[1,5-a]pyrimidine derivatives as potent CDK2 inhibitors for anticancer therapy.
    Article Snippet: Targeting Cyclin-Dependent Kinase 2 (CDK2) remains a critical strategy in anticancer drug discovery.. This study unveils a highly promising series of novel [1,2,4]triazolo[1,5-a]pyrimidine (TP) derivatives, achieved through innovative S/N-glycerolylation and peptide conjugation strategies.. We report the rational design, efficient multistep synthesis (yields up to 85 %), and comprehensive biological and computational evaluation.

    Purification:

    Article Title: A Simplified Strategy for Nanobody Production and Use Based on Functional GST-Nanobody Fusion Proteins
    Article Snippet: .. The buffer of purified recombinant GFP, GST-nb-GFP, mCherry, and GST-nb-mCherry was exchanged into ITC buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, pH 7.4) using 7K MWCO ZebaTM spin desalting columns (ThermoFisher Scientific). .. ITC measurements were carried out at 25 ◦C using a MicroCal PEAQ-ITC instrument (Malvern Panalytical, Worcestershire, United Kingdom).

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    Article Snippet: .. The buffer of purified recombinant GFP, GST-nb-GFP, mCherry, and GST-nb-mCherry was exchanged into ITC buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, pH 7.4) using 7K MWCO Zeba TM spin desalting columns (ThermoFisher Scientific). .. ITC measurements were carried out at 25 °C using a MicroCal PEAQ-ITC instrument (Malvern Panalytical, Worcestershire, United Kingdom).

    Protein Concentration:

    Article Title: FragLite mapping to identify the BRD4 recruitment site of P-TEFb
    Article Snippet: Samples were excited using a wavelength of 337 nm and emission spectra measured at 620 nm and 665 nm (PHERAstar FS (BMG LABTECH)). .. CDK9-cyclin T2 was buffer exchanged using a HiTrap desalting column (5 mL) (Cytiva) into ITC buffer (50 mM HEPES, 300 mM NaCl, 0.5 mM TCEP, pH 7.4) and protein concentration was then determined using a Nanodrop 2000 at an absorbance of 280 nm with sequence derived extinction coefficients ( http://web.expasy.org/ protparam/). ..

    Sequencing:

    Article Title: FragLite mapping to identify the BRD4 recruitment site of P-TEFb
    Article Snippet: Samples were excited using a wavelength of 337 nm and emission spectra measured at 620 nm and 665 nm (PHERAstar FS (BMG LABTECH)). .. CDK9-cyclin T2 was buffer exchanged using a HiTrap desalting column (5 mL) (Cytiva) into ITC buffer (50 mM HEPES, 300 mM NaCl, 0.5 mM TCEP, pH 7.4) and protein concentration was then determined using a Nanodrop 2000 at an absorbance of 280 nm with sequence derived extinction coefficients ( http://web.expasy.org/ protparam/). ..

    Derivative Assay:

    Article Title: FragLite mapping to identify the BRD4 recruitment site of P-TEFb
    Article Snippet: Samples were excited using a wavelength of 337 nm and emission spectra measured at 620 nm and 665 nm (PHERAstar FS (BMG LABTECH)). .. CDK9-cyclin T2 was buffer exchanged using a HiTrap desalting column (5 mL) (Cytiva) into ITC buffer (50 mM HEPES, 300 mM NaCl, 0.5 mM TCEP, pH 7.4) and protein concentration was then determined using a Nanodrop 2000 at an absorbance of 280 nm with sequence derived extinction coefficients ( http://web.expasy.org/ protparam/). ..



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    ( a ) Characterisation of BRD4 mutants that disrupt binding to CDK9-cyclin T1. Mutation of residues Gln1350, Leu1354 and Phe1357, highlighted by the FragLite map and AlphaFold3 model disrupt BRD4 binding to CDK9-Cyclin T1. ( b ) Mutation of BRD4 residues Leu1354 and Phe1357, leads to a loss of BRD4 binding to CDK9-cyclin T2 in a fluorescence polarisation (FP) assay. ( c ) The cyclin T1 Tyr175Ala mutation reduces the Homogenous Time-Resolved Fluorescence (HTRF) signal, whereas the Trp210Ala mutation, previously identified as important for AFF4 interaction and adjacent to Tyr175, shows signals comparable to wild-type. ( d-g ) Isothermal Titration Calorimetry (ITC) plots of CDK9-cyclin T2 complexes with BRD4. Representative titration plots for (d) CDK9-cyclin T2 (e) CDK9-cyclin T2 Tyr174Ala, (f) CDK9-cyclin T2 Phe175Ala (negative control), and (g) CDK9-cyclin T2 Trp206Ala vs the BRD4 P-TEFb Interaction Domain (PID). The top panels show the raw heat signal, and the bottom panels show the integrated heat per injection fitted to a single-site binding model. HTRF experiments were carried out in triplicate and repeated on three separate days. The error bars indicate SD. FP experiments were carried out in triplicate and repeated on three separate days. ITC thermodynamic parameters (K d ) were derived from three independent biological replicates. Derived K d values are compiled in . Related to and Supplementary Figure 11.

    Journal: bioRxiv

    Article Title: FragLite mapping to identify the BRD4 recruitment site of P-TEFb

    doi: 10.64898/2026.04.09.717428

    Figure Lengend Snippet: ( a ) Characterisation of BRD4 mutants that disrupt binding to CDK9-cyclin T1. Mutation of residues Gln1350, Leu1354 and Phe1357, highlighted by the FragLite map and AlphaFold3 model disrupt BRD4 binding to CDK9-Cyclin T1. ( b ) Mutation of BRD4 residues Leu1354 and Phe1357, leads to a loss of BRD4 binding to CDK9-cyclin T2 in a fluorescence polarisation (FP) assay. ( c ) The cyclin T1 Tyr175Ala mutation reduces the Homogenous Time-Resolved Fluorescence (HTRF) signal, whereas the Trp210Ala mutation, previously identified as important for AFF4 interaction and adjacent to Tyr175, shows signals comparable to wild-type. ( d-g ) Isothermal Titration Calorimetry (ITC) plots of CDK9-cyclin T2 complexes with BRD4. Representative titration plots for (d) CDK9-cyclin T2 (e) CDK9-cyclin T2 Tyr174Ala, (f) CDK9-cyclin T2 Phe175Ala (negative control), and (g) CDK9-cyclin T2 Trp206Ala vs the BRD4 P-TEFb Interaction Domain (PID). The top panels show the raw heat signal, and the bottom panels show the integrated heat per injection fitted to a single-site binding model. HTRF experiments were carried out in triplicate and repeated on three separate days. The error bars indicate SD. FP experiments were carried out in triplicate and repeated on three separate days. ITC thermodynamic parameters (K d ) were derived from three independent biological replicates. Derived K d values are compiled in . Related to and Supplementary Figure 11.

    Article Snippet: CDK9-cyclin T2 was buffer exchanged using a HiTrap desalting column (5 mL) (Cytiva) into ITC buffer (50 mM HEPES, 300 mM NaCl, 0.5 mM TCEP, pH 7.4) and protein concentration was then determined using a Nanodrop 2000 at an absorbance of 280 nm with sequence derived extinction coefficients ( http://web.expasy.org/ protparam/).

    Techniques: Binding Assay, Mutagenesis, Fluorescence, FP Assay, Isothermal Titration Calorimetry, Titration, Negative Control, Injection, Derivative Assay