transition to adulthood for youth with developmental disabilities
chemical thermodynamics - basic concepts and methods
Ngày tải lên :
02/04/2014, 16:43
... derivative to relate it to other quantities that are readily available The key to obtaining an expression for a particular partial derivative is to start with the total derivative for the dependent ... (c) calorie Calculate the conversion factor for changing atmosphere to pascal and atmosphere to bar 2.2 Calculate the conversion factor for changing calorie to (a) cubic meter atmosphere and (b) ... principles We have taken the opportunity, therefore, to revise our approach to some topics and to add problems that reflect new applications We have continued to take advantage of the resources available...
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basic statistics understanding conventional methods and modern insights jul 2009
Ngày tải lên :
11/06/2014, 00:51
... goal here is to illustrate what might go wrong and provide information about where to look for better techniques First we consider a situation where the histogram tends to perform tolerably well ... but less than or equal to −20, and so on Determine the frequencies for each bin and construct a histogram For the data in table 2.2, suppose a histogram is to be created with bins defined as follows: ... customary to omit the index of summation That is, simply use the notation Xi = X1 + X2 + · · · + Xn Example Imagine you work for a software company and you want to know, when customers call for...
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development of novel micro-embossing methods and microfluidic designs for biomedical applications
Ngày tải lên :
14/11/2014, 13:26
... requirements for a fabrication process due to the presence of proteins and even cells on the device For example, protein or antibody needs to be pre-loaded onto the channel surface before bonding ... Whitesides, 1998] combined a photolithography technique with PDMS molding for microfabrication The PDMS resin was cast onto a photoresist mold produced by photolithography on a silicon wafer ... Electron beam lithography was performed to directly pattern on PMMA The basic idea is to select two different polymers with different glass transition temperatures with higher Tg polymer being the...
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Glycoprotein Methods and Protocols - P1
Ngày tải lên :
08/10/2012, 10:25
... show a tendency to adhere to Sephacryl gels, which is not seen with Sepharose gels 2.4 Ion-Exchange High-Performance Liquid Chromatography Ion-exchange high performance liquid chromatography is carried ... the running buffer to the column through an injector Monitor the eluate on-line with an ultraviolet (UV) monitor and collect fractions using a fraction collector and subject to the appropriate ... injector Monitor the eluate on-line with a UV monitor and collect fractions using a fraction collector and subject to the appropriate carbohydrate and antibody analyses 3.5 Ion-Exchange Chromatography...
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Glycoprotein Methods and Protocols - P2
Ngày tải lên :
08/10/2012, 10:25
... water for and then rinse in distilled water Treat sections with Schiff’s reagent for 10–30 Wash for 10 in running tap water Counterstain with Mayer’s hematoxylin for to Wash in running tap water for ... and incubate for 30 to h Proceed with histochemistry protocol as usual 3.2.3 Enzymatic Deglycosylation to Confirm Lectin Specificity See Subheadings 3.6.1.–3.6.4 Histological Methods for Detection ... or the nature of the sugars substituted with sulfate For this information, we must turn to lectin histochemistry and immunohistochemistry 1.1.2 Lectin Histochemistry Lectins are a diverse group...
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Glycoprotein Methods and Protocols - P3
Ngày tải lên :
08/10/2012, 10:25
... antibody to control for nonspecific binding Incubation buffers need to contain protein (50% of blocking concentration) and/or nonionic detergent Incubate for 1–24 h at 4–37°C with agitation To save ... (or streptavidin-peroxidase for biotin) will also need to be optimized to give the best signal -to- background ratio Affinity-purified antibodies with crossreactivity with other species' antibodies ... example, a core protein epitope– specific antibody can be used for capture and then several antibodies reactive with different carbohydrate epitopes can be used for detection to both quantitate and...
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Glycoprotein Methods and Protocols - P4
Ngày tải lên :
08/10/2012, 10:25
... (down to µm) for histological processing the better, because this provides clearer details of cell structure It may be necessary, in some cases, to use thicker sections (up to 20 µm) in order to ... for studies in humans is given in Newton et al (30) This histological and fixation method can be adapted to staining the adherent mucous gel with biotinylated antibodies and lectins coupled to ... stored at –20°C For smaller gut segments, e.g., rat, a good method is to leave the stomach and intestine intact, ligate one end, fill with 0.9% NaCl, and ligate the other end before snap-freezing...
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Glycoprotein Methods and Protocols - P5
Ngày tải lên :
08/10/2012, 10:25
... apparatus and air-dry for Perform all ensuing procedures in Blotto as follows: a Incubate in Blotto for 30 min, and incubate with antibody directed against nonglycosylated peptide epitopes of the mucin ... mucin of interest for 90 b Wash two times for 15 each c Incubate with 0.5 µCi of (185 kBq) 125I-labeled protein A for 60 d Wash twice for each in Blotto, and then wash once for in phosphatebuffered ... mucins is seldom quantitative, owing to the large amount of stored yet unlabeled mucin that competes with the labeled mucin for the antibody (see also Chapter 20) Fortunately, it appears that the...
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Glycoprotein Methods and Protocols - P6
Ngày tải lên :
08/10/2012, 10:25
... Leave to cool before pouring (hand hot) and insert well-forming comb (see Note 2) Leave to set for at least h prior to use 3.3.2.3 ELECTROPHORESIS Electrophorese sample in electrophoresis buffer for ... evaporator Add 50 µL of redrying buffer to each sample and dry, under vacuum, for 30 Add 50 µL of freshly prepared coupling buffer to each sample and vortex to mix well Leave at room temperature for ... Thornton et al 3.3.1 Anion-Exchange Chromatography The flow rate for anion-exchange is 0.5 mL/min throughout the chromatography, and typically 0.5-mL fractions are collected Apply the sample (up to...
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Glycoprotein Methods and Protocols - P7
Ngày tải lên :
08/10/2012, 10:25
... (40,000 rpm) for 2.5 h at 15°C in a Beckman SW40 Ti swing-out rotor The gradient was emptied from the top into 0.5-mL fractions, and these were analyzed for reactivity with antisera for the MUC5AC ... swing-out rotor) for desired time at 15°C (in our case 2.5 h) (see Note 8) Unload tubes from the top with a pipet (for a gradient of this volume we usually take 0.5-mL fractions) Analyze fractions with ... 1.3 mL/min/tube (see Note 6) Weigh tubes to check that they contain equal volumes Carefully apply sample (up to 500 µL) with a Hamilton syringe to the top of the gradient (see Note 7) Centrifuge...
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Glycoprotein Methods and Protocols - P8
Ngày tải lên :
08/10/2012, 10:25
... adjusted to pH 8.5 with NaOH Buffer may be kept up to mo at 4°C Fmoc reagent: mg/mL fluorenylmethyl chloroformate (Sigma, cat no F0378) in acetonitrile (HPLC grade) Reagent may be stored up to wk ... vessel in a 105°C oven for 24 h Remove the vessel from the oven and open the vacuum tap immediately within a fume hood (see Note 4) Place the vials into a vacuum centrifuge for 10 to evaporate excess ... derivatized with 9-fluorenylmethyl chloroformate J Chromatogr 723, 219–225 Wilkins, M R., Yan, J X., and Gooley, A A (1999) 2-DE spots amino acid analysis with 9-fluorenylmethyl chloroformate, 2-D...
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Glycoprotein Methods and Protocols - P9
Ngày tải lên :
08/10/2012, 10:25
... methods for the separation of PTH-glycosylated amino acids on a modified Beckman LF3600 sequenator (12) These methods were developed for two reasons: (1) to provide a distinct elution window for ... glycosylated peptides substituted with large oligosaccharides it may be necessary to treat with TFMSA (13) or glycosidases before sequencing There are two principle reasons for desialylating glycopeptides: ... of coupling to ~5% 14 Amine buffers such as Tris interfere with coupling to arylamine and should be avoided 15 The EDC reagent should be mixed with the reaction buffer just prior to coupling...
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Glycoprotein Methods and Protocols - P10
Ngày tải lên :
08/10/2012, 10:25
... corresponding to the MUC1 VNTR sequence synthesized according to the Pepscan approach to epitope mapping Antibodies are allowed to react with each peptide, and those containing the epitope or minimum ... as follows: a DMF to cover gears for b MeOH (complete submersion) for c MeOH to cover gears for (three times) The pins are then allowed to air-dry in an acid-free fume hood for a minimum of 30 ... of the epitope and may also provide the forces between epitope and paratope required to maintain binding Conversely, it is possible to identify residues that are present in the epitope but can...
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Glycoprotein Methods and Protocols - P11
Ngày tải lên :
08/10/2012, 10:25
... and 6, and continue the PCR with 30 cycles at 94˚C for 30 s, 50˚C for and 72˚C for min, ending with a 72˚C extension for 148 Bell and Forstner Check the PCR product for size and purity by electrophoresis ... culture incubator for 43 h Remove serum-containing DMEM and wash COS cells thoroughly with DMEM without serum Replace medium with mL of DMEM without serum and incubate at 37˚C for another 24 ... the Forstner laboratory, Dr Gongqiao Xu, and Dr Ismat Khatri DNA sequencing was performed by the Biotechnology Service Centre, The Hospital for Sick Children, Toronto, Canada References Forstner,...
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Glycoprotein Methods and Protocols - P12
Ngày tải lên :
08/10/2012, 10:25
... acids react with periodate under acidic conditions to produce β-formylpyruvic acid, which condenses with TBA to produce a purple chromogen (λmax = 549 nm) The assay is sensitive to nmol, but ... glycoprotein corresponding to 10 µg of total sugars with 100 µL of M TFA in the presence of µg of mesoinositol used as internal standard at 100°C for h in glass tubes fitted with a Teflon screw cap ... be separated without introduction of base in the eluent Because the detector works only at high pH, postcolumn addition of NaOH to the effluent before the entry into the PAD detector is required...
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Glycoprotein Methods and Protocols - P13
Ngày tải lên :
08/10/2012, 10:25
... onto disk and leave for 15 (see Note 15) Wash the tube with 100 µL acetonitrile and add to disk Wash disk with mL of acetonitrile followed by × mL 4% water in acetonitrile Elute the sample with ... 75% at 60 for the total run 3.6.2 Porous Graphitized Carbon Chromatography Wash the column with water for 30 at a flow rate of 0.75 mL/min (see Note 9) Wash the column with acetonitrile for 30 at ... Elute the sample with the following gradient at a flow rate of 0.75 mL/min with water/ acetonitrile containing 0.2% TFA: a 20% acetonitrile for b 40% acetonitrile over 33 c 30% acetonitrile over...
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Glycoprotein Methods and Protocols - P14
Ngày tải lên :
08/10/2012, 10:25
... spectroscopy (COSY) provides information on directly coupled protons with regard to coupling constants Starting from the anomeric proton, the H-2, H-3, H-4, and so on, atom resonances, which were ... III 1H-4/13C-4) For the β-Gal units, the 13C-atom resonances 2II, 3II, 2III, and 4III are deshielded from to 10 ppm, as compared to the standard values observed for β-methylgalactoside On the contrary, ... the diagonal Two-dimensional total correlation spectroscopy (2D 1HTOCSY) can be used to characterize all the proton resonances A transfer of magnetization from H-1 to H-6 is observed in the case...
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Glycoprotein Methods and Protocols - P15
Ngày tải lên :
08/10/2012, 10:25
... µg Our system for sulfate analysis uses the same chromatographic system as that for sugar analysis, in which the ion chromatography column and high-performance anion-exchange chromatography (HPAEC) ... mercaptoethanol) Ideally, desalting into water should be performed as a matter of course prior to sulfate analysis For proteins that have poor solubility in water, consider desalting into partially ... a 100°C heating block and hydrolyze for precisely h After briefly centrifuging to spin condensate to the bottom of the tube, transfer the hydrolyzed samples to a vacuum centrifuge and dry completely...
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Glycoprotein Methods and Protocols - P16
Ngày tải lên :
08/10/2012, 10:25
... cells with 60 Kgray (6 mrad) of radiation, or treat with 10 µg/mL of mitomycin C (Sigma) for h Wash the cells and produce a single-cell suspension using a Pasteur pipet (wide aperture to avoid ... Conditioned medium: DMEM is supplemented with 10% FBS, mM glutamine, 100 IU/mL of penicillin, 100 µg/mL of streptomycin, and put onto 24-h postconfluent 3T3 cell layers for 24 h After conditioning, the ... 100 µg/mL of streptomycin, 3.7 g/L of NaHCO3, and nonessential amino acids (sterile 100X stock solution, Gibco/ BRL), supplemented with 20% of FBS (LS174T cells) or 10% of FBS for Caco-2 and A431...
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