The purified periplasmic domain of ToxR (ToxR-C) was used for the Isothermal Titration Calorimetry (ITC) analysis. ITC measurements were carried out using an automated ITC200 (MicroCal, GE Healthcare Life Sciences, Piscataway, NJ). The reference cell was filled with buffer (20 mM Na2HPO4 pH 7.8 and 1 mM EDTA). The reaction cell was filled with 0.2 mM of the periplasmic domain of ToxR, and a syringe was filled with either 10 mM cFP or 10 mM linear phenylalanine-proline dipeptide (FP). Two microliters of either cFP or FP was injected into the reaction cell containing ToxR-C at a concentration of 0.2 mM until saturated. Titrations were performed to give a series of 2 μl injections at 2 min intervals with 1,000 rpm stirring at 25 ° C. As a control, cFP was titrated into the reference buffer (20 mM Na2HPO4 pH 7.8 and 1 mM EDTA). Data was evaluated in terms of a simple binding model using the ORIGIN software package (OriginLab, Northampton, MA).
Origin software package
Manufactured by OriginLab
Sourced in United States
Origin is a data analysis and graphing software package. It provides tools for data acquisition, data analysis, and visualization. The software can be used to plot and analyze data from various sources.
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Lab products found in correlation
Pymol software, by Schrödinger (1 mentions)
Uv 1800 spectrophotometer, by Shimadzu (1 mentions)
Microcal vp itc calorimeter, by Malvern Panalytical (1 mentions)
Nano dsc, by TA Instruments (1 mentions)
Vp dsc microcal microcalorimeter, by Malvern Panalytical (1 mentions)
Varian 2000 ftir scimitar spectrometer, by Agilent Technologies (1 mentions)
Golden gate single reflection diamond atr accessory, by Specac (1 mentions)
16 protocols using origin software package
1
ITC Analysis of ToxR Periplasmic Domain
2
Phytoextraction Capacity Assessment Metrics
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The BCF (bioconcentration factor) values, TF (translocation factor) values, and uptake amounts (amounts of HMs of shoots), were calculated to identify performance indicators for assessing the plant phytoextraction capacity. The calculation method of these indicators used the following formulas:
where Cshoot is the concentration of Pb (or Tl) in plant dry aboveground biomass (mg kg−1, DW); Csoil is the concentration of Pb (or Tl) in the soil (mg kg−1, DW); DWshoot is the dry aboveground biomass (g, DW); and Croot is the concentration of Pb (or Tl) in dry belowground biomass (mg kg−1, DW).
Mapping was performed using the Origin software package (version 8.5; OriginLab, Northampton, MA, USA). Statistical and linear regression analyses were performed using GraphPad Prism version 6 (GraphPad Software, Inc., California, USA). Duncan’s multiple range test in SPSS 17 (SPSS Inc., Chicago, IL, USA) was used to compare the effects of different treatment schemes at a 0.05 significance level. Data were expressed as means plus orminus one standard deviation.
where Cshoot is the concentration of Pb (or Tl) in plant dry aboveground biomass (mg kg−1, DW); Csoil is the concentration of Pb (or Tl) in the soil (mg kg−1, DW); DWshoot is the dry aboveground biomass (g, DW); and Croot is the concentration of Pb (or Tl) in dry belowground biomass (mg kg−1, DW).
Mapping was performed using the Origin software package (version 8.5; OriginLab, Northampton, MA, USA). Statistical and linear regression analyses were performed using GraphPad Prism version 6 (GraphPad Software, Inc., California, USA). Duncan’s multiple range test in SPSS 17 (SPSS Inc., Chicago, IL, USA) was used to compare the effects of different treatment schemes at a 0.05 significance level. Data were expressed as means plus orminus one standard deviation.
3
Comparative Analysis of Experimental Conditions
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All the parameters were measured on 3 replicates and were expressed as the mean. Statistical analysis was performed using the Origin software package (version 9.0; OriginLab Cor., Hampton). Differences between effects were assessed by the Tukey test (P < 0.05).
4
Kaplan-Meier Survival Analysis
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We used the Origin software package (OriginLab Corporation, Massachusetts, USA), to carry out statistical analysis and to determine lifespan values. The Kaplan Meier estimator was used to evaluate differences between survivals and determine the p-values.
5
Identification and Characterization of DmaHsp70s
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Open reading frames (ORF) of DmaHsp70s were identified by NCBI ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html ). Identifications of the cDNA clones were determined by the BLAST search in GenBank (http://www.ncbi.nlm.nih.gov/BLAST ). The molecular weights (Mw) and isoelectric point (pI) values were calculated by SMS2 server (http://www.detaibio.com/sms2/ ) and presented with Origin software package (version 9.8.0.200, Origin Lab, Massachusetts, USA). The subcellular localization of DmaHsp70s were predicted by WoLF PSORT online server (https://wolfpsort.hgc.jp ). SMART server (SMART: Main page (embl.de)) was employed to identify the conserved domains of DmaHsp70s using default parameters. Characteristic sequences of DmaHsp70s were identified by Prosite (https://prosite.expasy.org/ ). The 3D structure of the DmaHsp70s were simulated by homology modeling (https://swissmodel.expasy.org/ ). PyMOL software (version 2.4, Schrödinger, New York, USA) was used to visualize the 3D structures. DmaHsp70s were aligned respectively with Hsp70s from other species in Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/ ) using default setting and presented by the DNAMAN9 software package (Lynnon Corporation, Pointe-Claire, Quebec, Canada).
6
Thermal Stability Analysis of Reaction Centers
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Pigment extraction and pigment composition analysis of RCs were performed as previously described [18 (link)]. Thermal stability was investigated at 48 °C according to previous methodology [19 (link)], with the difference that 0.1% LDAO (Sigma-Aldrich, St. Louis, MO, USA) was used as detergent instead of 0.1% Triton X-100. The number of intact RCs in the sample was estimated by the absorption of monomeric BChl at 804 as reported [9 (link)]. The construction of the curves of absorption changes was carried out using the Origin software package (OriginLab Corporation, Northampton, MA, USA). Differential (light minus dark) absorption spectra were recorded at constant illumination with SZS-22 and KS-19 crossed light filters using a Shimadzu UV-1800 spectrophotometer (Shimadzu Corporation, Kyoto, Japan).
7
Kinetics of dsRNA Cleavage by Dcr-2
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Kinetics of dsRNA cleavage and siRNA release were investigated by monitoring the first round of catalytic cycle for ~5 min. The reaction mixture was synchronized at the beginning of the catalytic cycle via rapid mixing using the stopped-flow system, and by using 10-fold excess of dmDcr-2 over dsRNA. The excess enzyme over substrate ensured that dsRNA remained bound during the first turnover, thus making precise measurement of kinetic parameters feasible. dsRNA used in the cleavage assay had a FRET pair at the primary cleavage site, and cleavage and siRNA release were recorded by exciting at 530 nm (Cy3 excitation) and monitoring the time-dependent change in FRET signal (Cy5 fluorescence emission) using 670 nm long pass filter. Assays were performed in cleavage assay buffer (25 mM Tris pH 8.0, 100 mM KCl, 10 mM MgCl2.6H2O, 1 mM TCEP) at 25°C in the absence and presence of nucleotide (ATP/ATPγS). Nucleotide-bound dmDcr-2 (dmDcr-2•ATP/ATPγS) was prepared by incubating 2 μM of dmDcr-2 with 8 mM ATP/ ATPγS for 5 min. At least 4–10 traces were obtained and values averaged. Averaged traces were analyzed using single or double exponential rate equations, and data analysis was performed using the Origin software package (OriginLab corporation).
8
Biophysical Characterization of Protein-Ligand Interactions
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ITC data was recorded in a MicroCal VP-ITC calorimeter (Malvern). All proteins analyzed by ITC were subjected to a final purification step by size exclusion chromatography in ITC buffer (25 mM HEPES, 100 mM KCl, 0.5 mM TCEP, pH 7.5). Titrations were performed at 25°C and consisted of an initial 2 μl injection followed by a series of 28 injections of 10 μl each. In the cases where an interaction was detected a blank run was performed by titrating the titrant into ITC buffer. Data were processed with the ORIGIN software package (OriginLab) and in cases where an interaction was detected either the blank run was subtracted from the initial isotherm (to correct for the heat of dilution) or data were further processed for global fitting with the NITPIC, SEDPHAT, and GUSSI software packages.
9
Curve Fitting for APV Data
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The APV data were not perfectly smooth because of instrument error related to the use of the VHX-1000E Digital Microscope, which affected the calculation accuracy of the hydration degree (α). Therefore, the Origin software package (OriginLab Corporation) was used to fit curves to the plots of the APVs for the three color channels (r, g, and b) versus time. The fitted curves and their equations are shown in Figure 4 and Table 3 , respectively.
10
Comparative Statistical Analysis Protocol
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Results are expressed as mean±standard error of the mean (SEM) unless stated otherwise. One-way ANOVA followed by a Tukey-Kramer multiple comparisons test, and two-way ANOVA followed by a Bonferroni multiple comparisons test were performed; p<0.05 was considered significant. All statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software Inc., San Diego, CA, USA) or Origin software package version 5.0 (Origin Lab Corporation, Northampton, MA, USA).
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