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1、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 t
2、he 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-str
3、anded (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-D
4、NA 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 cle
5、ar 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
6、- 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 interacts with histones H3 and H4 but not H2A and H2B,Results:1)
7、 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 f
8、actor 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 piece
9、s,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
10、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: DNas
11、e 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
12、 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 interactio
13、n 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
14、 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 - Pr
15、otein 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.,NT012 - Protein Interaction in Different Buffer Systems,Fig. 2: Binding of NT-647-labeled TEM1 to BLIP p
16、rotein. A) Binding of BLIP-WT to NT-647-labeled TEM1-WT. A Kd of 3.5 nM 0.6 nM was determined for this interaction. B) Binding of BLIP-W112A to NT-647-labeled TEM1-WT. A Kd of 474 nM 76 nM was determined for this interaction. C) Binding of BLIP1-W150A to NT-647-labeled TEM1-WT. A Kd of 1750 nM 220 nM was determined for this interaction.,NT012 - Protein Interaction in Different Buffer Systems,Fig. 3: binding of Ypet-BLIP to TEM1 protein. A) Binding of Ypet-BLIP-WT to TEM1-WT. A Kd of 4.7 nM 0.7
