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Supplementary Materials and Method Immunostaining and Western Blot
Supplementary Materials and Method Immunostaining and Western Blot Analysis For immunofluorescence staining, mouse and human cells were fixed with 4% paraformaldehyde- PBS for 15 min. Following Triton-X100 permeabilization and blocking, cells were incubated with primary antibodies overnight at 4°C following with Alexa 594-conjugated secondary antibodies at 4°C for 1 hour (Thermo Fisher Scientific, 1:1000). Samples were mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories) and immunofluorescence was detected using Olympus confocal microscopy. For western blot analysis, cells were lysed on ice using RIPA buffer supplemented with protease and phosphatase inhibitors (Sigma). Primary Antibodies for Immunostaining and Western Blot Analysis: Yap (14074, Cell Signaling), pYAP (4911, Cell Signaling), Lats1 (3477, Cell Signaling), pLats1( 8654, Cell Signaling), Wnt5a (2530, Cell Signaling), cleaved Caspase-3 (9661, Cell Signaling), Ki-67 (VP-K451, Vector Laboratories), Cyr61 (sc-13100, Santa Cruz Biotechnology), CTGF (sc-14939, Santa Cruz Biotechnology), AXL (8661, Cell Signaling), pErk (4376, Cell Signaling), pMEK (4376, Cell Signaling), Ck-19 (16858-1-AP, Proteintech), Actin (A2228, Sigma Aldrich), Vinculin (V4139, Sigma Aldrich), Kras (sc-30, Santa Cruz Biotechnology). Ectopic expression of YAP1 and WNT5A in mouse and human cells To generate YAP1S127A-expressing stable Pa04C cells, Pa04C cells were transfected with a linearized pcDNA3.1 plasmid with or without YAP1 cDNA containing S127A substitution. Two days post-transfection using Lipofectamine1000, cultures were selected in G418 (Sigma) and single clones were picked and expanded for further analysis. Overexpression of YAPS127A or WNT5A in human or mouse cells other than Pa04C were acheieved with lentivral infection. Briefly, lentivirus infection was performed by transfecting 293T cells with either GFP control, YAP1S127A, or WNT5A cloned in pHAGE lentivirus vector {EF1α promoter-GW-IRES-eGFP (GW: Gateway modified)}. -
Green Fluorescent Protein (GFP) Purification Student Manual
Green Fluorescent Protein (GFP) Purification Student Manual "Bioengineered DNA was, weight for weight, the most valuable material in the world. A single microscopic bacterium, too small to see with the human eye, but containing the gene for a heart attack enzyme, streptokinase, or for "ice-minus" which prevented frost damage to crops, might be worth 5 billion dollars to the right buyer." Michael Crichton - Jurassic Park Contents Lesson 1 Genetic Transformation Review—Finding the Green Fluorescent Molecule Lesson 2 Inoculation—Growing a Cell Culture Lesson 3 Purification Phase 1—Bacterial Concentration and Lysis Lesson 4 Purification Phase 2—Removing Bacterial Debris Lesson 5 Purification Phase 3—Protein Chromatography 26 Lesson 1 Finding the Green Fluorescent Molecule Genetic Transformation Review In Bio-Rad Kit 1, you performed a genetic transformation of E. coli bacterial cells. The results of this procedure were colonies of cells that fluoresced when exposed to ultraviolet light. This is not a normal phenotype (characteristic) for E.coli. You were then asked to fig- ure out a way to determine which molecule was becoming fluorescent under UV light. After determining that the pGLO plasmid DNA was not responsible for the fluorescence under the UV light, you concluded that it was not the plasmid DNA that was fluorescing in response to the ultraviolet light within the cells. This then led to the next hypothesis that if it is not the DNA fluorescing when exposed to the UV light, then it must be a protein that the new DNA pro- duces within the cells. 1. Proteins. a. What is a protein? b. -
Protein Structure & Folding
6 Protein Structure & Folding To understand protein folding In the last chapter we learned that proteins are composed of amino acids Goal as a chemical equilibrium. linked together by peptide bonds. We also learned that the twenty amino acids display a wide range of chemical properties. In this chapter we will see Objectives that how a protein folds is determined by its amino acid sequence and that the three-dimensional shape of a folded protein determines its function by After this chapter, you should be able to: the way it positions these amino acids. Finally, we will see that proteins fold • describe the four levels of protein because doing so minimizes Gibbs free energy and that this minimization structure and the thermodynamic involves both making the most favorable bonds and maximizing disorder. forces that stabilize them. • explain how entropy (S) and enthalpy Proteins exhibit four levels of structure (H) contribute to Gibbs free energy. • use the equation ΔG = ΔH – TΔS The structure of proteins can be broken down into four levels of to determine the dependence of organization. The first is primary structure, the linear sequence of amino the favorability of a reaction on acids in the polypeptide chain. By convention, the primary sequence is temperature. written in order from the amino acid at the N-terminus (by convention • explain the hydrophobic effect and its usually on the left) to the amino acid at the C-terminus. The second level role in protein folding. of protein structure, secondary structure, is the local conformation adopted by stretches of contiguous amino acids. -
Developing Back Reflectance Absorbance As a Useful Technique
Design of a Simple Cryogenic System for Ultraviolet-Visible Absorption Spectroscopy with a Back-reflectance Fiber Optic Probe Andrew Vinyard, Kaj Hansen, Ross Byrd, Douglas A Stuart * and John Hansen * Department of Chemistry, University of West Georgia *Corresponding Authors, email: [email protected] and [email protected] Abstract We report a convenient and inexpensive technique for the rapid acquisition of absorption spectra from small samples at cryogenic temperatures using a home built cryostat with novel collection optics. A cylindrical copper block was constructed with a coaxial bore to hold a 4.00 mm diameter EPR tube and mounted on a copper feed in thermal contact with liquid nitrogen. A 6.35 mm diameter hole was bored into the side of the cylinder so a fiber optic cable bundle could be positioned orthogonally to the EPR tube. The light passing through the sample is reflected off of the opposing surfaces of the EPR tube and surrounding copper, back through the sample. The emergent light is then collected by the fiber optic bundle, and analyzed by a dispersive spectrometer. Absorption spectra for KMnO4 were measured between 400 nm and 700 nm. Absorption intensity at 506 nm, 525 nm, 545 nm and 567 nm was found to be proportional to concentration, displaying Beer’s law like behavior. The EPR tube had an internal diameter of 3.2 mm; the double pass of the probe beam through the sample affords a central path length of about 6.4 mm. Comparing these measurements with those recorded on a conventional tabletop spectrometer using a cuvette with a 10.00 mm path length, we consistently found a ratio between intensities of 0.58 rather than the anticipated 0.64. -
Protocols and Tips in Protein Purification
Department of Molecular Biology & Biotechnology Protocols and tips in protein purification or How to purify protein in one day Second edition 2018 2 Contents I. Introduction 7 II. General sequence of protein purification procedures 9 Preparation of equipment and reagents 9 Preparation and use of stock solutions 10 Chromatography system 11 Preparation of chromatographic columns 13 Preparation of crude extract (cell free extract or soluble proteins fraction) 17 Pre chromatographic steps 18 Chromatographic steps 18 Sequence of operations during IEC and HIC 18 Ion exchange chromatography (IEC) 19 Hydrophobic interaction chromatography (HIC) 21 Gel filtration (SEC) 22 Affinity chromatography 24 Purification of His-tagged proteins 25 Purification of GST-tagged proteins 26 Purification of MBP-tagged proteins 26 Low affinity chromatography 26 III. “Common sense” strategy in protein purification 27 General principles and tips in “common sense” strategy 27 Algorithm for development of purification protocol for soluble over expressed protein 29 Brief scheme of purification of soluble protein 36 Timing for refined purification protocol of soluble over -expressed protein 37 DNA-binding proteins 38 IV. Protocols 41 1. Preparation of the stock solutions 41 2. Quick and effective cell disruption and preparation of the cell free extract 42 3. Protamin sulphate (PS) treatment 43 4. Analytical ammonium sulphate cut (AM cut) 43 5. Preparative ammonium sulphate cut 43 6. Precipitation of proteins by ammonium sulphate 44 7. Recovery of protein from the ammonium sulphate precipitate 44 8. Analysis of solubility of expression 45 9. Analysis of expression for low expressed His tagged protein 46 10. Bio-Rad protein assay Sveta’s easy protocol 47 11. -
DNA Glycosylase Exercise - Levels 1 & 2: Answer Key
Name________________________ StarBiochem DNA Glycosylase Exercise - Levels 1 & 2: Answer Key Background In this exercise, you will explore the structure of a DNA repair protein found in most species, including bacteria. DNA repair proteins move along DNA strands, checking for mistakes or damage. DNA glycosylases, a specific type of DNA repair protein, recognize DNA bases that have been chemically altered and remove them, leaving a site in the DNA without a base. Other proteins then come along to fill in the missing DNA base. Learning objectives We will explore the relationship between a protein’s structure and its function in a human DNA glycosylase called human 8-oxoguanine glycosylase (hOGG1). Getting started We will begin this exercise by exploring the structure of hOGG1 using a molecular 3-D viewer called StarBiochem. In this particular structure, the repair protein is bound to a segment of DNA that has been damaged. We will first focus on the structure hOGG1 and then on how this protein interacts with DNA to repair a damaged DNA base. • To begin using StarBiochem, please navigate to: http://mit.edu/star/biochem/. • Click on the Start button Click on the Start button for StarBiochem. • Click Trust when a prompt appears asking if you trust the certificate. • In the top menu, click on Samples à Select from Samples. Within the Amino Acid/Proteins à Protein tab, select “DNA glycosylase hOGG1 w/ DNA – H. sapiens (1EBM)”. “1EBM” is the four character unique ID for this structure. Take a moment to look at the structure from various angles by rotating and zooming on the structure. -
2 in 1 Fluorescence and Absorbance Spectrometer
2 in 1 Fluorescence and Application Fluorescence Absorbance Spectrometer: Note How does it work? Life Sciences Figure 1: Duetta (left) and the inside of the Duetta sample compartment (right) with direction of the light path Introduction Duetta™ is a 2-in-1 fluorescence and absorbance excitation energy and the spectrum at longer wavelengths spectrometer from HORIBA Scientific. There are several is acquired to measure the distribution of intensity and benefits of having two different spectroscopies in one energy (wavelength) of the photons emitted. instrument. For high concentration solutions of interest, both primary and secondary inner-filter effects will affect Fluorescence Excitation Spectrum the fluorescence spectrum measured on a standard Acquiring the intensity of photons emitted at a single fluorometer. Having absorbance and fluorescence emission wavelength and scanning the excitation spectroscopy on the same instrument enables Duetta monochromator to excite the population of molecules in and EzSpec software to apply corrections for inner-filter the sample with different wavelengths. A fluorescence effect and provide more accurate data, for a wider range of excitation spectrum is analogous to an absorbance sample concentrations. Another benefit of the two in one spectrum, but is specific to a single emitting species/ instrument is that absorbance results and fluorescence wavelength as opposed to collecting all absorbing species results contain less error since the sample does not move in a sample or solution. from one instrument to another to get both measurements. Standard fluorescence methods such as Emission %Transmittance Spectrum Spectrum and Excitation Spectrum are available on Duetta, This is a ratio, in terms of percentage, of the intensity of but EzSpec software gives a user the ability to measure transmitted light through an absorbing sample (I) compared the Absorbance Spectrum as a stand-alone method or to the transmitted light through a blank solvent (I0). -
Structural Analysis of 3′Utrs in Insect Flaviviruses
bioRxiv preprint doi: https://doi.org/10.1101/2021.06.23.449515; this version posted June 24, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Structural analysis of 3’UTRs in insect flaviviruses reveals novel determinant of sfRNA biogenesis and provides new insights into flavivirus evolution Andrii Slonchak1,#, Rhys Parry1, Brody Pullinger1, Julian D.J. Sng1, Xiaohui Wang1, Teresa F Buck1,2, Francisco J Torres1, Jessica J Harrison1, Agathe M G Colmant1, Jody Hobson- Peters1,3, Roy A Hall1,3, Andrew Tuplin4, Alexander A Khromykh1,3,# 1 School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, Australia 2Current affiliation: Institute for Medical and Marine Biotechnology, University of Lübeck, Lübeck, Germany 3Australian Infectious Diseases Research Centre, Global Virus Network Centre of Excellence, Brisbane, QLD, Australia 4 School of Molecular and Cellular Biology, University of Leeds, Leeds, U.K. # corresponding authors: [email protected]; [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.23.449515; this version posted June 24, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Insect-specific flaviviruses (ISFs) circulate in nature due to vertical transmission in mosquitoes and do not infect vertebrates. ISFs include two distinct lineages – classical ISFs (cISFs) that evolved independently and dual host associated ISFs (dISFs) that are proposed to diverge from mosquito-borne flaviviruses (MBFs). Compared to pathogenic flaviviruses, ISFs are relatively poorly studied, and their molecular biology remains largely unexplored. -
Structural Mechanisms of Oligomer and Amyloid Fibril Formation by the Prion Protein Cite This: Chem
ChemComm View Article Online FEATURE ARTICLE View Journal | View Issue Structural mechanisms of oligomer and amyloid fibril formation by the prion protein Cite this: Chem. Commun., 2018, 54,6230 Ishita Sengupta a and Jayant B. Udgaonkar *b Misfolding and aggregation of the prion protein is responsible for multiple neurodegenerative diseases. Works from several laboratories on folding of both the WT and multiple pathogenic mutant variants of the prion protein have identified several structurally dissimilar intermediates, which might be potential precursors to misfolding and aggregation. The misfolded aggregates themselves are morphologically distinct, critically dependent on the solution conditions under which they are prepared, but always b-sheet rich. Despite the lack of an atomic resolution structure of the infectious pathogenic agent in prion diseases, several low resolution models have identified the b-sheet rich core of the aggregates formed in vitro, to lie in the a2–a3 subdomain of the prion protein, albeit with local stabilities that vary Received 17th April 2018, with the type of aggregate. This feature article describes recent advances in the investigation of in vitro Accepted 14th May 2018 prion protein aggregation using multiple spectroscopic probes, with particular focus on (1) identifying DOI: 10.1039/c8cc03053g aggregation-prone conformations of the monomeric protein, (2) conditions which trigger misfolding and oligomerization, (3) the mechanism of misfolding and aggregation, and (4) the structure of the misfolded rsc.li/chemcomm intermediates and final aggregates. 1. Introduction The prion protein can exist in two distinct structural isoforms: a National Centre for Biological Sciences, Tata Institute of Fundamental Research, PrPC and PrPSc. -
Giri Narasimhan ECS 254A; Phone: X3748 [email protected] July 2011
BSC 4934: QʼBIC Capstone Workshop" Giri Narasimhan ECS 254A; Phone: x3748 [email protected] http://www.cs.fiu.edu/~giri/teach/BSC4934_Su11.html July 2011 7/19/11 Q'BIC Bioinformatics 1 Modular Nature of Proteins" Proteins# are collections of “modular” domains. For example, Coagulation Factor XII F2 E F2 E K Catalytic Domain F2 E K K Catalytic Domain PLAT 7/21/10 Modular Nature of Protein Structures" Example: Diphtheria Toxin 7/21/10 Domain Architecture Tools" CDART# " #Protein AAH24495; Domain Architecture; " #It’s domain relatives; " #Multiple alignment for 2nd domain SMART# 7/21/10 Active Sites" Active sites in proteins are usually hydrophobic pockets/ crevices/troughs that involve sidechain atoms. 7/21/10 Active Sites" Left PDB 3RTD (streptavidin) and the first site located by the MOE Site Finder. Middle 3RTD with complexed ligand (biotin). Right Biotin ligand overlaid with calculated alpha spheres of the first site. 7/21/10 Secondary Structure Prediction Software" 7/21/10 PDB: Protein Data Bank" #Database of protein tertiary and quaternary structures and protein complexes. http:// www.rcsb.org/pdb/ #Over 29,000 structures as of Feb 1, 2005. #Structures determined by " # NMR Spectroscopy " # X-ray crystallography " # Computational prediction methods #Sample PDB file: Click here [▪] 7/21/10 Protein Folding" Unfolded Rapid (< 1s) Molten Globule State Slow (1 – 1000 s) Folded Native State #How to find minimum energy configuration? 7/21/10 Protein Structures" #Most proteins have a hydrophobic core. #Within the core, specific interactions take place between amino acid side chains. #Can an amino acid be replaced by some other amino acid? " # Limited by space and available contacts with nearby amino acids #Outside the core, proteins are composed of loops and structural elements in contact with water, solvent, other proteins and other structures. -
Caenorhabditis Elegans BRICHOS Domain–Containing Protein C09F5.1 Maintains Thermotolerance and Decreases Cytotoxicity of A42 B
G C A T T A C G G C A T genes Article Caenorhabditis elegans BRICHOS Domain–Containing Protein C09F5.1 Maintains Thermotolerance and Decreases Cytotoxicity of Aβ42 by Activating the UPR Myungchul Song 1, Kyunghee Song 1,2, Sunghee Kim 1,3, Jinyoung Lee 1,4, Sueyun Hwang 5 and Chingtack Han 1,* 1 Department of Life Science, Sogang University, Seoul 04107, Korea; [email protected] (M.S.); [email protected] (K.S.); [email protected] (S.K.); jinylee@amorepacific.com (J.L.) 2 LG Household & Health Care, Daejeon 34114, Korea 3 Department of Medicine, Biomedical Research Institute, Seoul National University Hospital, Seoul 03080, Korea 4 Amorepacific R&D Center, Yongin 17074, Korea 5 Department of Chemical Engineering, Hankyung National University, Anseong 17579, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-2-705-8454 Received: 11 December 2017; Accepted: 9 March 2018; Published: 13 March 2018 Abstract: Caenorhabditis elegans C09F5.1 is a nematode-specific gene that encodes a type II transmembrane protein containing the BRICHOS domain. The gene was isolated as a heat-sensitive mutant, but the function of the protein remained unclear. We examined the expression pattern and subcellular localization of C09F5.1 as well as its roles in thermotolerance and chaperone function. Expression of C09F5.1 under heat shock conditions was induced in a heat shock factor 1 (HSF-1)–dependent manner. However, under normal growth conditions, most cells types exposed to mechanical stimuli expressed C09F5.1. Knockdown of C09F5.1 expression or deletion of the N-terminal domain decreased thermotolerance. -
Modified Bradford Assay Method of Protein Quantification Utilising Dye Reagents from Four Nigerian Plants
International Journal of Research Studies in Biosciences (IJRSB) Volume 3, Issue 12, December 2015, PP 79-87 ISSN 2349-0357 (Print) & ISSN 2349-0365 (Online) www.arcjournals.org Modified Bradford Assay Method of Protein Quantification Utilising Dye Reagents from Four Nigerian Plants *S. O. Okeniyi+, *J. Ogbodobri, **A. O. Oyedeji, *P. E. Omale, *M. M. Adeyemi, *S. Garba, ***J. A. Lori *Department.of Chemistry, Nigerian Defence Academy, Afaka, Kaduna State - Nigeria ** Department of Chemical & Physical Sciences, Walter Sisulu University Eastern Cape, South Africa ***Department of Chemistry, Bingham University, Karu, Nassarawa State - Nigeria Abstract: Aqueous and organic solvents extraction process using ethanol, methanol and chloroform were carried out with four different Nigerian plants namely: Pterocarpus osun (uhe), Lawsonia inermis (lalle), Bixa Orellana (annatto) and Hibiscus sabderriffa (zobo) to extract dye reagents from the plants. The ability of the dye reagents to replace Coomassie Brilliant Blue in the Bradford assay method of protein quantification were determined and compared. The solvents extracts gave good colourful results in the extraction of the dye reagents while only aqueous extract of Hisbiscus sabderiffa (zobo) gave similar results to that of solvent extracts. The solvent extracts obtained from Pterocarpus osun (uhe), Lawsonia inermis (lalle) and Bixa Orellana (annatto) plants could not be used to estimate amino acids from protein samples. However, solvent extracts of Hibiscus sabderriffa (zobo) was able to estimate amino acids from protein samples. The change in maximum wavelength (λmax) and the increased absorption with zobo dye reagent; on addition of protein samples showed that solvent extract of Hibiscus sabderiffa (zobo) dye has the potential to quantify and estimate amino acids in protein samples as much as the Coomassie Blue utilised in the Bradford assay method.