br Experimental design materials and

Experimental design, materials and methods
Mobility Retardation of NCALD (and other NCS proteins) in native gels has been documented and the retardation was directly dependent on the concentration of calcium [1,2]. Electrophoreses in native gels and analyses were carried out as described [1]. Extraneous calcium in solutions was removed through treatment with Chelex-100 resin (BioRad Laboratories, CA, USA) using standard procedures. In order to determine if divalent cations such as calcium were still carried over in solutions, ICP analyses were carried out. Components other than the protein and calcium calibration buffer in the sample loaded for electrophoresis are: 20mM Tris pH 7.5 (Chelex-treated), loading dye (63mM Tris HCl, 0.1%, betamercaptoethanol, 0.0005% bromophenol blue, 10% Glycerol) and running buffer (25mM Tris, 192mM glycine). The concentration of calcium in these components was determined by ICP. Chelex-treated water served as a blank. Briefly, samples were diluted into 10mL of 5% nitric buy NSC59984 in ICP grade water rated at 18.2MΩcm. The quantity of calcium present was estimated as parts per billion (ppb) and converted into molarity. The data is presented as a table. It is noted that 20mM Tris pH 7.5 (Chelex-treated) and the loading dye were comparable to Chelex-water, which served as the reference. Only the running buffer contained measurable calcium above the reference. Binding of magnesium to two NCS proteins, GCAP1 and GCAP2, with functional consequences has been demonstrated [3,4]. Therefore, the concentration of magnesium in these components was also determined by ICP. Based on the determination, the loaded sample in the native gel experiments contained 0.4169±0.0024μM magnesium. However, addition of magnesium (even up to 400μM) has no effect on any tested NCS protein [1,5].
Acknowledgements The work was supported by grants from the New Jersey Health (previously UMDNJ) Foundation and the Osteopathic Heritage Foundation. The support by the RowanSOM Graduate School of Biomedical Sciences is also acknowledged. Mr. Andrew Shore is gratefully acknowledged for his help with the ICP measurements.
1. Specifications table
2. Value of the data
3. Data The forward SILAC analysis quantified 1459 proteins whereas reverse SILAC analysis quantified 1712 proteins. However, a total of 390 proteins were reproducibly identified and quantified in both forward and reverse reactions using a highly stringent data analysis protocol (see Supplementary data Table, Excel® file). String (version 9.1) analysis was deployed to analyse the co-expression pattern of all significantly expression-changed proteins [1].
4. Experimental design, materials & methods
Acknowledgments Saifur R. Khan (supervised by Arno G. Siraki) is supported by an Alberta Innovates Technology Futures (AITF) Graduate Student Scholarship. This work was supported by the Canadian Institutes of Health Research (202034) and the Natural Sciences and Engineering Research Council of Canada (#RGPIN-2014-04878).
Specifications Table
Value of the data
Experimental design, materials and methods
Acknowledgments This research was supported by the USDA-NIFA HATCH Grant number 13-3110006050, and also by NIH Grant numbers 5 P30 RR032135 from the COBRE Program of the National Center for Research Resources and 8 P30 GM 103498 from the National Institute of General Medical Sciences. The Vermont Genetics Network Proteomics Facility is supported through NIH grant P20GM103449 from the INBRE Program of the National Institute of General Medical Sciences. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH.
Specifications Table
Value of data
1. Data The data were generated by two sets of all-atoms MD simulations involving the hCES1 structure in complex with both Amplex Red (AR) and the corresponding enzymatic product deacetylAR. The first set involved standard 5ns MD runs with a view (a) to assess the stability of the hCES1-AR complex and (b) to reveal the egress process for the enzymatic product (at least in its initial phase). The second set involved steered MD runs (SMD) in order to offer a quantitative comparison of the stability of the two simulated complexes and to reveal the energy factors governing the undocking processes.