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Mechanisms of Fibril Formation and Cellular Response
Published in Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin, XIth International Symposium on Amyloidosis, 2007
Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin
Isolation and Characterization of Amyloid Protein: Leptomeninges dissected from the ventral pons and brainstem were hand homogenized in 0.1 M sodium citrate, 0.15 M sodium chloride, and centrifuged eight times. The final pellet was dialyzed versus water and lyophilized. The fibrils were solubilized with 6 M guanidine hydrochloride containing dithiothreitol, alkylated with iodoacetic acid, and centrifuged (4). The supernatant was fractionated on a Sepharose CL6B column and pooled fractions were exhaustively dialyzed against water and lyophilized. Pools were analyzed by SDS-PAGE using a Tris-Tricine gel system. Pool 4
Suppression of Transthyretin Synthesis by Antisense Oligonucleotides
Published in Gilles Grateau, Robert A. Kyle, Martha Skinner, Amyloid and Amyloidosis, 2004
M.D. Benson, B. Kluve-Beckerman, K.W. Sloop, D.M. Bodenmiller
Mice were injected with a TTR specific anti-sense oligonucleotide, random oligonucleotide, or saline twice weekly for four weeks. Blood samples were collected once each week. TTR was separated from other serum proteins by Tris-Tricine SDS-PAGE and then quantified by scanning densitometry. TTR values were expressed relative to the TTR signal in week 1 serum of saline-treated mouse. The four values whithin each condition were determined from weekly bleeds of the same mouse.
The Modification of Lysine
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
Urabe and co-workers51 prepared various mixed carboxylic acid anhydrides of tetradecanoic acid and oxa derivatives which varied in their “hydrophobicity”. This represented an attempt to change the surface properties of the enzyme molecule, in this case, thermolysin. The carboxylic acid anhydrides were formed in situ from the corresponding acid and ethyclhloroformate in dioxane with triethylamine. The modification reaction was performed in 0.013 M barbital, 0.013 M CaCl2, pH 8.5 containing 39% (v/v) dioxane and was terminated with neutral hydroxylamine which also served to remove O-acyl derivatives. The extent of reaction was determined by titration with trinitrobenzenesulfonic acid. Derivatives obtained with tetradecanoic acid and 4-oxatetradecanoic acid were insoluble. Derivatives obtained with 4,7,10-trioxatetradecanoic acid and 4,7,10,13-tetraoxotetradecanoic acid both had approximately seven amino groups modified per mole of enzyme, showed little if any loss in either proteinase or esterase activity, and possessed enhanced thermal stability. Howlett and Wardrop52 were able to dissociate the components of human erythrocyte membrane by the use of 3,4,5,6-tetrahydrophthalic anhydride. The reaction was performed in 0.02 M Tricine, pH 8.5. The 3,4,5,6-tetrahydrophthalic anhydride was introduced into the reaction mixture as a dioxane solution (a maximum of 0.10 ml/5 ml reaction mixture). The pH was maintained at pH 8.0 to 9.0 with 1.0 M NaOH. The reaction was considered complete when no further change in pH was observed. The extent of modification was determined by titration with trinitrobenzenesulfonic acid. The reaction could be reversed by incubation for 24 to 48 h at ambient temperature following the addition of an equal volume of 0.1M potassium phosphate, pH 5.4 (the final pH of the reaction mixture was 6.0).
The successful strategy of comprehensive pre-implantation genetic testing for beta-thalassaemia–haemoglobin E disease and chromosome balance using karyomapping
Published in Journal of Obstetrics and Gynaecology, 2022
Sirivipa Piyamongkol, Suchada Mongkolchaipak, Pimlak Charoenkwan, Rungthiwa Sirapat, Wanwisa Suriya, Tawiwan Pantasri, Theera Tongsong, Wirawit Piyamongkol
Biopsied cells were washed thoroughly in phosphate-buffered saline (PBS, Cell Signaling Technology, Theera Trading Co. Ltd., Bangkok, Thailand) with 0.1% polyvinyl alcohol (PVA, Sigma-Aldrich, Chiangmai VM Co., Ltd., Chiang Mai, Thailand) before transference to microcentrifuge tubes. DNA extraction was performed using an alkaline lysis buffer protocol (Sermon et al. 1995). 2.5 μL of lysis buffer (0.75 μL of water, 1.25 μL of 0.1 M DTT and 0.5 μL of 1 M NaOH) was added, mixtures were incubated at 60 °C for 10 min. After that, a neutralisation buffer (2.5 μL of 0.4 M tricine) was added. Whole genome amplification with multiple displacement amplification (MDA, REPLI-g® Single Cell Kit, Chiangmai VM Co., Ltd., Chiang Mai, Thailand) was then carried out by manufacturer’s instructions. A mixture of 12.5 μL of water, 29 μL of reaction buffer and 1 μL of DNA polymerase (REPLI-g® Single Cell Kit) was added to extracted DNA, making a total volume of 50 μL. The mixtures were incubated at 30 °C for 2 h then at 65 °C for 5 min to inactivate the reaction.
Evaluation of chromosomal aberrations induced by 188Re-dendrimer nanosystem on B16f1 melanoma cells
Published in International Journal of Radiation Biology, 2018
Marcos Tassano, Natalia Oddone, Marcelo Fernández, Williams Porcal, María Fernanda García, Wilner Martínez-López, Juan Claudio Benech, Pablo Cabral
The radioactive labeling of the conjugate was performed by two steps: 10 mg tricine (N-[Tris (hydroxymethyl) methyl] glycine, Sigma, St. Louis, MO) was dissolved in 0.8 mL of distilled water pH 4.5–5. In a second vial, 15 mg of SnCl2·2H2O (JT Baker, Phillipsburg, NJ) was dissolved in 0.5 mL 2.0 M HCl and 0.05 mL of this solution was added to the tricine solution (solution A). In order to perform radiolabeling, 25 mL of solution A was added to 10 mg of purified dendrimer-HYNIC. An activity up to 37 MBq (1 mCi) of 188ReO4− (obtained from 188W/188Re generator, POLATOM, Otwock, Poland) was added to the mixture and incubated at 50 °C for 60 min. The radiolabeled compound was purified by gel filtration with Sephadex G-25 column (PD-10 Sephadex G-25, Pharmacia, Peapack, NJ). Labeling yield, purity and stability were performed by HPLC (high performance liquid chromatography) with gamma and UV detector (Agilent Technologies Inc., 1200 Infinity Series equipped with GABI Star detector, Santa Clara, CA). Utilizing an C18 column (MCH-10, 150 × 4.6 mm), with mobile phases of ultrapure water with TFA (2,2,2-trifluoroacetic acid) 0.14% (solvent A) and ACN (acetonitrile) with 0.14% TFA (solvent B), with a flow of 1 mL/min with a gradient of 0–30 min 100% solvent B.
Noninvasive evaluation of HABP1 expression with 99mTc-labeled small-interference RNA in ovarian cancer
Published in International Journal of Radiation Biology, 2021
Chunyu Duan, Yue Gao, Sha Luan, Shibo Guo, Xueliang Cao, Peng Xu, Peng Fu, Changjiu Zhao
The HYNIC-siHABP1 and HYNIC-NCsiRNA were labeled with 99mTc in the presence of Tricine as a co-ligand. Briefly, a solution of 10 μg HYNIC–siRNA in 50 μL bicarbonate buffer (25 mM, pH 8.5) was added to 100 μL Tricine aqueous solution (7 mg/mL), followed by 74 MBq 99mTCO4- and 10 μL SnCl2 in 0.1 M HCl (1 mg/mL). The mix was incubated at RT for 60 min and purified with Sep-Pak C18 (Waters). Radioactivity and absorbance at 260 nm of the purified solution were determined. Labeling efficiency and radiochemical purity were measured by HPLC (GX-281, Gilson, France) with a C18 column (10 μm, 19 × 250 mm, Waters). The mobile phase was 30% acetonitrile aqueous solution at a flow rate of 1 mL/min at RT.