China
Africa
Explore chapters and articles related to this topic
Basic Molecular Cloning of DNA and RNA
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
Another ubiquitous application of site-directed mutagenesis is the targeted insertion of cysteines, which have a reactive –SH group. In this case, it is not key amino acids of the protein that are targeted but, rather, key structural regions, as the idea is not to disrupt function but to insert a tag. After the protein is expressed, a tag bearing an alkylating agent, such as a maleimide, iodoacetamide, or similar agent, will react with the thiol group and form a covalent bond. (These are discussed more fully in Chapters 7 and 14.) Thiol-reactive compounds bearing fluorescent dyes, luminescent probes, spin probes, or other tags can be added to proteins expressing inserted cysteine(s) and will attach to the specified location(s) (Figure 2.18). The biggest caveat with this technique is that it is necessary to ensure that the cysteine-substituted protein has normal function before it can be used as a model of the wild type. For some proteins, such as ion channels, measurement of function can be relatively straightforward (e.g., current–voltage relationships); for other proteins, this is more difficult.
Synthetic Polymer-Drug Conjugates for Human Therapy
Published in Vladimir Torchilin, Mansoor M Amiji, Handbook of Materials for Nanomedicine, 2011
Thiol group-containing drugs can be conjugated with PEG bearing maleimide, 2-pyridyldisulfanyl, iodoacetamide or vinyl sulfone groups162 (Fig. 1.6) forming linkages stable or degradable in reducing environment of the cell compartments.
Mass spectrometry techniques for detection of COVID-19 viral and host proteins using naso-oropharyngeal swab and plasma
Published in Sanjeeva Srivastava, Multi-Pronged Omics Technologies to Understand COVID-19, 2022
In this section, we will discuss the detailed workflow of immune–affinity-based plasma depletion. The detailed description of the sample preparation method is shown in Table 3.1. A 2-ml of whole-blood sample is collected in a sterile vacutainer by clinicians. The blood samples are centrifuged at 3,000 rpm for 10 minutes. The plasma samples are then collected carefully from the top of the centrifuged tube. These samples are then heated at 56°C for 30 minutes for viral inactivation. The samples are aliquoted into several vials and stored at −80°C until further use. The depletion of high abundant proteins such as fibrinogen, haptoglobin, albumin, apolipoprotein A-I, and others are carried out using the PierceTM 12 abundant protein depletion spin column (ThermoFisher Scientific). Around 15 µl of plasma samples were mixed with the resin of the spin column and incubated under a rocking motion for 1 hour. Around 500 µl samples were eluted by centrifugation 1,500 g for 2 minutes and concentrated to 100 µl using SpeedVac vacuum. The quantification of depleted plasma samples was carried out by Bradford assay taking bovine serum albumin (BSA) protein as a reference standard. To 30 µg of a depleted plasma sample was added 6 M of urea. The plasma protein was reduced with 20 mM tris(2-carboxyethyl) phosphine (TCEP) and incubated at 37°C for 1 hour. The alkylation was carried out by 40 mM iodoacetamide (IAA) and incubated for 15 minutes in the dark. Then, the sample was diluted six times with 50 mM ammonium bicarbonate (ABC). The proteins were digested using trypsin at an enzyme/substrate ratio of 1:30 and incubated overnight at 37°C. Next day, the digested sample was vacuum dried to stop trypsin activity and reconstituted in 0.1% formic acid (FA). The digested peptide was desalted using a C-18 column, and the eluted dried peptides were reconstituted in 0.1% (v/v) FA. Finally, the peptide concentration was calculated from its OD value at 205 and 280 nm using the Scopes method (Scopes 1974). Around 1 µg of peptides was injected at a flow rate of 300 nl/minute into liquid chromatography (LC) and the peptides were analyzed on Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Fischer Scientific) with an easy nano LC-1200 system (Suvarna et al. 2021).
Proteomic analysis of secretomes from Bacillus sp. AR03: characterization of enzymatic cocktails active on complex carbohydrates for xylooligosaccharides production
Published in Preparative Biochemistry & Biotechnology, 2021
Johan S. Hero, José H. Pisa, Enzo E. Raimondo, M. Alejandra Martínez
Protein preparation for LC-MS/MS analysis was performed according to La Greca et al.[14] with slight modifications. Briefly, 50 µg of protein from each condition were treated with 20 μL of reducing solution (200 mM DTT, 100 mM Tris, pH 7.8) and with alkylation solution (200 mM iodoacetamide, 100 mM Tris, pH 7.8) for 1 hr at room temperature. Then, proteins were precipitated with 10% TCA and centrifuged (4 °C, 16,000 × g, 30 min). Pellets were washed three times with pre-cooled acetone (−20 °C), resuspended in 50 mM ammonium bicarbonate (pH 8.0) at a concentration of 10 µg µL−1, and digested with trypsin. Finally, the digested proteins were purified with a Zip-Tip C18 column, freeze-dried by Speed Vac, and preserved until their MS analysis.
Proteomic analysis of whole-body responses in medaka (Oryzias latipes) exposed to benzalkonium chloride
Published in Journal of Environmental Science and Health, Part A, 2020
Young Sang Kwon, Jae-Woong Jung, Yeong Jin Kim, Chang-Beom Park, Jong Cheol Shon, Jong-Hwan Kim, June-Woo Park, Sang Gon Kim, Jong-Su Seo
2-DE and image analysis were performed according to the method previously described by Kwon et al. (2016).[23] For first-dimension separation, quantified proteins (400 μg) were applied to 17 cm linear gradient pH 4–7 rehydrating IPG strips (Bio-Rad) and rehydrated passively for 12 h at room temperature. Isoelectric focusing (IEF) was performed in a Protean IEF cell (Bio-Rad) at 20 °C using a five-step program consisting of 30 min at 250 V, 1 h at 1,000 V, 1 h at 2,500 V, 1 h at 5,000 V, and 8 h at 8,000 V. The IPG strips were equilibrated (reduction and alkylation) by incubating them for 15 min in equilibration buffer comprised of 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, and trace bromophenol blue containing 1% DTT for reduction and 2.5% iodoacetamide for alkylation. In the second dimension (2-DE), strips were transferred to the top of a pre-cast Criterion 11% SDS polyacrylamide gel and samples were run simultaneously on all gels by SDS-PAGE for 2 h at 150 V using a Protean Xi Dodeca Cell (Bio-Rad). The gels were stained for 24 h with 0.1% (w/v) Coomassie Brilliant Blue G250, and de-stained for 4 h in deionized water. 2-DE gel images were digitized using a GS-800 Imaging Densitometer (Bio-Rad) with Quantity One software (Bio-Rad), and image analysis was performed with PDQuest version 7.2.0 software (Bio-Rad). For each sample, quantitative analysis was performed with individual replicates of the analytical gel. Proteins with a statistically significant difference between the control and BAC-treated groups (P < 0.05 by a one-way ANOVA test) were selected for identification.
L-asparaginase from Aspergillus oryzae spp.: effects of production process and biochemical parameters
Published in Preparative Biochemistry & Biotechnology, 2022
Marília Crivelari da Cunha, Jessika Gonçalves dos Santos Aguilar, Santa Maria Del Rosário Orrillo Lindo, Ruann Janser Soares de Castro, Helia Harumi Sato
L-cysteine and iodoacetamide decreased 15.6% and 28.8% of the enzymatic activity of L-asparaginase from A. oryzae IOC 3999, respectively. The sulfhydryl and thiol groups play an important role in preserving the enzyme structure, catalytic regulation, and electron transport.[44]