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Titelmasterformat durch Klicken bearbeiten,Textmasterformate durch Klicken bearbeiten,Zweite Ebene,Dritte Ebene,Vierte Ebene,Fnfte Ebene,#,Biomolecule Analytics,using Microscale Thermophoresis,Jiangsu University,Zhangyan,NT018-DNA-DNA Interaction Analysis,Title:,Thermodynamic characterization of DNA hybridization,Results:,(1)The binding,curves showed a typical sigmoidal shape,with the,ssDNA displaying a stronger MST response than the dsDNA.,(2)Increasing the,temperature resulted in a clear shift of the,Kd,towards higher values.,The,hybridization affinity was greatly reduced for the,mismatch-nucleotides.,Object:,Template ssDNA-Match ssDNA,Figure 1:A)Sequences of the DNA oligomers,B)Exemplary MST timetraces for a single-stranded(ssDNA)and a double-stranded DNA(dsDNA).,C)Fitted sigmoidal binding curve of a temperature-jump MST signal for a template-PM interaction.,D)Temperature-jump binding curves over a temperature range from 38,to 45,.,NT018-DNA-DNA Interaction Analysis,Figure 2:A)Vant Hoff plots of the DNA-DNA hybridization reactions.Template-PM in black,template-MM1 in blue and template-MM2 interactions in red.,B)Comparison between experimentally determined and calculated H and S,values(PM=perfect match,MM=mismatch).,Title:,The Decondensation factor 31 binds to mono-nucleosomes,Results:,1)A clear binding curve for the Df31 protein with mono-nucleosomes could be detected.However,BSA as control protein showed no binding to mono-nucleosomes.,2)The calculated,Kd from the measurements of the Df31 binding to mono-nucleosome was 3.72 0.56 M.,Object:,Decondensation factor 31-nucleosome,NT017-Characterization of a Protein Nucleosome Interaction,Fig.1 Measurement of Df31 binding to Cy5-labeled mono-nucleosomes.The Kd of the Df31-mono-nucleosome interaction was 3.72 0.56 M.,Title:,The Decondensation factor 31 specifically binds to ssRNA but not to ssDNA or dsDNA,Results:,1)A clear binding curve for the interaction of the Df31 protein and ssRNA could be detected.However,ssDNA and dsDNA showed no binding to Df31.,2)The calculated,Kd from the measurements of the Df31 binding to ssRNA was 24.01 3.15 M.,Object:,Decondensation factor 31-ssRNA/ssDNA/dsDNA,NT016-Characterization of Protein-Nucleic Acid Interactions,Fig.1 Measurement of Df31 binding to nucleic acids.The Kd of the Df31-ssRNA interaction was 24.01 3.15 M.,Title:,The Decondensation factor 31 specifically interacts with histones H3 and H4 but not H2A and H2B,Results:,1)A clear binding curve for the interaction of the Df31 protein and the core histones H3 and H4 could be detected.However,the core histones H2A and H2B showed no binding.,2)The calculated,Kd from the measurements of the Df31 binding to H3 was 1.5 0.14 M and H4 was 12 0.6 M.,Object:,Decondensation factor 31-Histones,NT015-Characterization of a Protein-Histone Interaction,Fig.1 Measurement of Df31-EGFP binding to core histones.The Kd of the Df31-H3 interaction was 1.5 0.15 M,the Kd of the Df31-H4 interaction was 12 0.6 M.,Title:,On a razors edge:watching Dnase I cutting DNA into pieces,Results:,1)DNase I requires Mg2+as a cofactor to cleave the phosphodiesterbond.If Mg2+be chelated by EDTA,Dnase I can not.,A dramatic change of thermophoresis can be assigned to DNase I nuclease activity and the digestion of the DNA substrate.,2)Using the quenching assay,we again observe a time dependent change in thermophoresis which can be assigned to DNase I nuclease activity.,Object:,Dnase I-DNA,NT014-Enzyme kinetics,Figure 1:Crystal structure of DNase I in complex with DNA,Figure 2:Schematic representation of DNase I nuclease activity.,NT014-Enzyme kinetics,Figure 3:DNase I endonuclease activity monitored by MST.,A.Time traces of DNase I activity were recorded with 1 s/1 s/1 s for IR Laser off/on/off.,B.Normalized,relative fluorescence change was plotted against time.Data were fit(red line)and the time constant was determined to 247 s(4 s).,Figure 4:DNase I endonuclease activity using a quenching approach.Normalized,relative fluorescence change was plotted against time.This result could be verified by two independent experiments.,Title:,Using MST to analyse the binding of the-Lactamase TEM1 to BLIP,Results:,1)The calculated,Kd for the interaction between WT TEM1 and WT BLIP was 3.5 nM+/-0.6 nM.,2),A 100-fold weaker binding affinity of NT-647-labeled TEM1-WT to BLIP-W112A.A mutation of tryptophan to alanine at position 150 resulted in a 600-fold weaker,Kd,as compared to WT BLIP.,3),A,Kd of 4.7 nM+/-0.75 nM was determined for the binding of Ypet-WT BLIP to WT TEM.,A 50-fold weaker affinity was determined for the binding of Ypet-BLIP-WT to TEM1-R243A.,4)Analyzed the binding of Ypet-BLIP-WT to TEM1-WT in mammalian cell lysates.,The,Kd was in the low nM range.,Object,:,-,Lactamase(TEM1),-Lactamase Inhibitory Protein(BLIP),NT012-Protein Interaction in Different Buffer Systems,Fig.1:Structural representation of the TEM1-BLIP complex.TEM1 is represented in cyan.BLIP is shown in white.TEM1 contact residues are in yellow.,NT0
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