DOCSLIB.ORG

Advances in Protein Chemistry Edited by Ghulam Md Ashraf Ishfaq Ahmed Sheikh Editors

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Advances in Protein Chemistry Edited by Ghulam Md Ashraf Ishfaq Ahmed Sheikh Editors www.esciencecentral.org/ebooks Advances in Protein Chemistry Edited by Ghulam Md Ashraf Ishfaq Ahmed Sheikh Editors Dr. Ghulam Md Ashraf (Email: [email protected]) Dr. Ishfaq Ahmed Sheikh (Email: [email protected]) King Fahd Medical Research Center King Abdulaziz University, P.O. Box 80216 Jeddah, Saudi Arabia Tyrosine Nitrated Proteins: Biochemistry and Pathophysiology Haseeb Ahsan* Department of Biochemistry, Faculty of Dentistry, Jamia Millia Islamia (A Central University), Okhla, New Delhi – 110025, India *Corresponding author: Dr. Haseeb Ahsan, Department of Biochemistry, Faculty of Dentistry, Jamia Millia Islamia (A Central University), Okhla, New Delhi – 110025, India, E-mail: drhahsan@ gmail.com Abstract The free radical-mediated damage to proteins results in the modification of amino acid residues, cross-linking of side chains and fragmentation. L-tyrosine and protein bound tyrosine are prone to attack by various mediators and reactive nitrogen intermediates to form 3-nitrotyrosine (3-NT). 3-NT formation is also catalyzed by a class of peroxidases utilizing nitrite and hydrogen peroxide as substrates. Evidence supports the formation of 3-NT in vivo in diverse pathologic conditions and 3-NT is thought to be a relatively specific marker of oxidative damage. The formation of nitrotyrosine represents a specific peroxynitrite-mediated protein modification; thus, detection of nitrotyrosine in proteins is considered as a biomarker for endogenous peroxynitrite activity. Formation of tyrosine nitrated proteins is considered to be a post-translational modification with important pathophysiological consequences and is one of the markers of nitrosative stress that have been reported in neurodegeneration, inflammatory and other pathological conditions. Introduction Most proteins contain tyrosine residues with a natural abundance of about 3% [1]. Tyrosine (one letter abbreviation, Y; three letter abbreviation, Tyr; also known as 4-hydroxyphenylalanine) is a non-essential amino acid and a member of the aromatic amino acid group. Tyrosine is mildly hydrophilic, a characteristic feature that is explained by the rather hydrophobic aromatic benzene ring carrying a hydroxyl group. As a consequence, tyrosine is often surface-exposed in proteins (only 15% of tyrosine residues are buried inside a protein) and therefore should be available for modification such as nitration by various factors [2-4]. Tyrosine can be modified by the addition of a nitro group (-NO2) in vivo with several agents resulting in the formation of 3-nitrotyrosine (3-NT) or tyrosine nitrated proteins (Figure 1). 3-NT [(2-Amino-3-(4-hydroxy-3-nitrophenyl) propanoic acid)] is a post-translational modification in proteins occurring through the action of a nitrating agent resulting in the addition of a -NO2 group (in ortho position + + NH3 NH3 COO- COO- RNS NO2 OH OH Tyrosine Nitrotyrosine (Tyr) (3-NT) Tyrosine decarboxylase Tyramine oxidase metabolism metabolism COO- COO- RNS NO2 OH OH p-hydroxyphenylacetic acid 3-nito-4-hydroxyphenylacetic acid (NHPA) (PHPA) OMICS Group eBooks Figure 1: Metabolism of tyrosine leading to the formation of products: p-hydroxyphenylacetic acid and 3-nitro-4-hydroxyphenylacetic acid (adapted 003 from Mani et al., 2003). to the phenolic hydroxyl group) leading to protein tyrosine nitration (PTN) [5]. Protein tyrosine nitration exhibits a certain degree of selectivity and not all tyrosine residues are nitrated since nitration may rather depend on the residue’s accessibility to solvents. Neither the abundance of a protein nor the number of tyrosine residues in a given protein can help us predict whether it is a target for PTN [2,3,6]. For example, human serum albumin (HSA) is less extensively nitrated than other plasma proteins, although being the most abundant plasma protein [6]. While HSA has 18 tyrosine residues, an in vitro study of peroxynitrite-mediated PTN showed that only two tyrosine residues are particularly susceptible to nitration [7]. The reactivity of a tyrosine residue might also depend on the nature of the reactive species [2]. While peroxynitrite (ONOO-) and tetranitromethane (TNM) nitrate certain proteins [8], there are differences in PTN patterns in other proteins [9-11]. The nitration of protein tyrosine residues could dramatically change protein structure and conformation and subsequently alter their function [12-15]. Tyrosine nitration sites are localized within specific functional domains of nitrated proteins [16]. For example, a strong inhibition of the catalytic activity of manganese-superoxide dismutase (MnSOD) by peroxynitrite-mediated PTN has been reported and explained by nitration of the essential tyrosine residue [17]. The inactivation of human MnSOD by peroxynitrite is caused by exclusive nitration of tyrosine 34 (Tyr34) to 3-nitrotyrosine [18]. Role of Free Radical Species in Tyrosine Nitration Nitrogen dioxide, nitrous acid, nitryl chloride, and certain peroxidases [19] derived from inflammatory cells can mediate the nitration of tyrosine to form 3-NT (Table 1). For example, nitrite (NO2-), a primary autoxidation product of NO [20], is further oxidized to form nitrogen dioxide by the action of peroxidases, e.g., myeloperoxidase and eosinophil peroxidase, heme proteins that are abundantly expressed in activated leukocytes. The resulting nitrogen dioxide nitrates the tyrosine residues in the presence of hydrogen peroxide (H2O2) [21]. Therefore, tyrosine nitration is based on the generation of nitrogen dioxide radicals (NO2) by various hemoperoxydases in the presence of H2O2 and nitrite [22-24]. Other plausible reactions are based on (i) the interaction of nitric oxide with a tyrosyl radical, (ii) the direct action of nitrogen dioxide, (iii) the formation of nitrous acid by acidification of nitrite, (iv) the oxidation of nitrite by hypochlorous acid to form nitryl chloride (NO2Cl), (v) the action of acyl or alkyl nitrates or (vi) the action of metal nitrates [1,25,26]. Hence, nitrotyrosine is therefore likely not a footprint for peroxynitrite alone but more generally a marker of nitrative stress. Nitrating Agent Proteins nitrated References Hemoperoxidases (myeloperoxidase, MPO lipoproteins peroxidase) – MPO/H2O2 system - Nitrite (NO2 ) hemoglobin Nitroprusside (nitropress) p65 (NF-κB) Tsikas (2012), Abello et al (2009). human serum albumin, creatine kinase, cytochrome c, hemoglobin, Peroxynitrite (ONO –) 2 NADH dehydrogenase, succinate dehydrogenase, H2A histone protein Tetranitromethane (TNM) bovine serum albumin, human serum albumin Table 1: Factors or agents that lead to the formation of tyrosine nitrated proteins. The reactive nitrogen species, peroxynitrite, is an important nitrating agentin vivo. However, it is not the only source of 3-NT formation in vivo. Initially, 3-NT was thought to be a biomarker of the existence of peroxynitrite, and protein tyrosine nitration was believed to be the biomarker of peroxynitrite formation in biological systems [27,28]. However, later studies demonstrated that some hemoproteins such as hemoglobin and myoglobin could catalyze NaNO2/H2O2-dependent nitration of tyrosine to yield 3-NT [29-31]. The possible underlying mechanism is that hemoproteins catalyze the oxidation of nitrite to nitrogen dioxide (NO2) which reacts with tyrosine radical (Tyr.) to form 3-NT [27,32,33]. Peroxynitrite and related reactive nitrogen species are capable of both oxidation and nitration of the aromatic side-chains of tyrosine and tryptophan in proteins [34,35], resulting in a condition known as “nitrosative stress”. Reactive oxygen species (ROS) and reactive nitrogen species (RNS)-mediated damage in the central nervous tissues may reflect an underlying neuroinflammatory process [36]. Protein damage that occurs under conditions of oxidative stress may represent direct oxidation of protein side-chains by ROS and/ or RNS or adduction of secondary products of oxidation of sugars (glycoxidation), or polyunsaturated fatty acids (lipid peroxidation) [36]. In addition to the well known or standard reactive oxygen and nitrogen species, oxidative damage to proteins can occur due to alternate oxidants (e.g., HOCl) and circulating oxidized amino acids such as tyrosine radical generated by metalloenzymes such as, myeloperoxidase [37]. The accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate ion of oxidized forms of proteins [38]. Carbon dioxide/bicarbonate (1.3 and 25 mM in plasma, respectively) [39] strongly influence peroxynitrite-mediated reactions [39- 43] and enhance nitration of aromatic rings as in tyrosine. They can also promote nitration in the presence of antioxidants (such as uric acid, ascorbate and thiols), which normally inhibit nitration, while partially inhibiting the oxidation of thiols. Carbon dioxide reacts - with peroxynitrite to form the nitrosoperoxycarbonate anion (ONOOCO2 ), which subsequently rearranges to form the nitrocarbonate - anion (O2NOCO2 ). The latter is considered to be the direct oxidant of peroxynitrite-mediated reagents in biological environments [43]. Peroxynitrite-mediated tyrosine nitration is also accelerated in the presence of transition metal ions, either in free form (Cu2+, Fe3+, Fe2+) or as complexes involving protoporphyrin IX (heme) or certain chelators - ethylene diamine tetraacetic acid (EDTA) [15,16,44]. Hence, metal ions catalysis plays an important role in the nitration of protein residues in proteins [45]. Role of Protein Tyrosine Nitration in
Load more

Recommended publications
  • Patterns of Green Fluorescent Protein Expression in Transgenic Plants
    Color profile: Generic CMYK printer profile Composite Default screen Plant Molecular Biology Reporter 18: 141a–141i, 2000 © 2000 International Society for Plant Molecular Biology. Printed in Canada. Publish by Abstract Patterns of Green Fluorescent Protein Expression in Transgenic Plants BRIAN K. HARPER1 and C. NEAL STEWART JR.2,* 1Novartis Agricultural Biotechnology Research, Inc., 3054 Cornwallis Rd., Research Triangle Park, NC 27709; 2Dept of Biology, University of North Carolina, Greensboro, NC 27402-6174 Abstract. Modified forms of genes encoding green fluorescent protein (GFP) can be mac- roscopically detected when expressed in whole plants. This technology has opened up new uses for GFP such as monitoring transgene presence and expression in the environment once it is linked or fused to a gene of interest. When whole-plant or whole-organ GFP vi- sualization is required, GFP should be predictably expressed and reliably fluorescent. In this study the whole plant expression and fluorescence patterns of a mGFP5er gene driven by the cauliflower mosaic virus 35S promoter was studied in intact GFP-expressing trans- genic tobacco (Nicotiana tabacum cv. Xanthi). It was shown that GFP synthesis levels in single plant organs were similar to GUS activity levels from published data when driven by the same promoter. Under the control of the 35S promoter, high expression of GFP can be used to visualize stems, young leaves, flowers, and organs where the 35S promoter is most active. Modified forms of GFP could replace GUS as the visual marker gene of choice. Key words: expression patterns, green fluorescent protein, marker genes Patterns of IntroductionGFP expression Harper and Stewart Since the discovery of green fluorescent protein (GFP) from the jellyfish Aequorea victoria it has become a frequently used tool in biology.
    [Show full text]
  • 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)}.
    [Show full text]
  • 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.
    [Show full text]
  • Protein Sequencing Production of Ions for Mass Spectrometry
    Lecture 1 Introduction- Protein Sequencing Production of Ions for Mass Spectrometry Nancy Allbritton, M.D., Ph.D. Department of Physiology & Biophysics 824-9137 (office) [email protected] Office- Rm D349 Medical Science D Bldg. Introduction to Proteins Amino Acid- structural unit of a protein α carbon Amino acids- linked by peptide (amide) bond Amino Acids Proteins- 20 amino acids (Recall DNA- 4 bases) R groups- Varying size, shape, charge, H-bonding capacity, & chemical reactivity Introduction to Proteins Polypeptide Chain (Protein) - Many amino acids linked by peptide bonds By convention: Residue 1 starts at amino terminus. Polypeptides- a. Main chain i.e. regularly repeating portion b. Side chains- variable portion Introduction to Proteins 25,000 human genes >2X106 proteins Natural Proteins - Typically 50-2000 amino acids i.e. 550-220,000 molecular weight Over 200 different types of post-translational modifications. Ex: proteolysis, phosphorylation, acetylation, glycosylation S S S S S S Ex: Insulin Protein Complexity Is Very Large Over 200 different types of post-translational modifications. The Problem of Protein Sequencing. Edman Degradation: Step-wise cleavage of an amino acid from the amino terminus of a peptide. Alanine Gly 1 2 Reacts with uncharged NH3 1 2 Gly 1 2 Cyclizes & Releases in Mild Acid H N-Gly 2 2 2 PTH-Alanine Edman Degradation 1. Must be short peptide (<50 a.a.) amino acid release- 98% efficiency proteins- must fragment (CNBr or trypsin) Trypsin 2. Frequently fails due to a blocked amino terminus 3. Intolerant of impurities 4. Tedious & time consuming (hours-days) 1 amino acid cycle- 2 hours Solution: Mass Spectrometry 1.
    [Show full text]
  • Analysis of Proteins by Immunoprecipitation
    Laboratory Procedures, PJ Hansen Laboratory - University of Florida Analysis of Proteins by Immunoprecipitation P.J. Hansen1 1Dept. of Animal Sciences, University of Florida Introduction Immunoprecipitation is a procedure by which peptides or proteins that react specifically with an antibody are removed from solution and examined for quantity or physical characteristics (molecular weight, isoelectric point, etc.). As usually practiced, the name of the procedure is a misnomer since removal of the antigen from solution does not depend upon the formation of an insoluble antibody-antigen complex. Rather, antibody-antigen complexes are removed from solution by addition of an insoluble form of an antibody binding protein such as Protein A, Protein G or second antibody (Figure 1). Thus, unlike other techniques based on immunoprecipitation, it is not necessary to determine the optimal antibody dilution that favors spontaneously-occurring immunoprecipitates. Figure 1. Schematic representation of the principle of immunoprecipitation. An antibody added to a mixture of radiolabeled (*) and unlabeled proteins binds specifically to its antigen (A) (left tube). Antibody- antigen complex is absorbed from solution through the addition of an immobilized antibody binding protein such as Protein A-Sepharose beads (middle panel). Upon centrifugation, the antibody-antigen complex is brought down in the pellet (right panel). Subsequent liberation of the antigen can be achieved by boiling the sample in the presence of SDS. Typically, the antigen is made radioactive before the immunoprecipitation procedure, either by culturing cells with radioactive precursor or by labeling the molecule after synthesis has been completed (e.g., by radioiodination to iodinate tyrosine residues or by sodium [3H]borohydride reduction to label carbohydrate).
    [Show full text]
  • 1 Supplementary Information in Vivo Mrna Display Enables
    Supplementary Information In vivo mRNA display enables Large-scale Proteomics by Next Generation Sequencing P. Oikonomou1,2,3, R. Salatino2, S. Tavazoie1,2,3* 1Department of Biological Sciences, Columbia University, New York City, New York, United States 2Department of Systems Biology, Columbia University, New York City, New York, United States 3Department of Biochemistry and Molecular Biophysics, Columbia University, New York City, New York, United States * Correspondence: P.O.: [email protected]; S.T.: [email protected] Table of Contents Methods ..................................................................................................................................... 4 Plasmid Construction .............................................................................................................. 4 Yeast Strains .......................................................................................................................... 4 In vivo mRNA display Library Generation ............................................................................... 5 Yeast cell culture .................................................................................................................... 5 Excess Coat Protein ............................................................................................................... 6 Non-Specific Functional Controls for in vivo mRNA display .................................................... 6 Whole cell lysate preparation .................................................................................................
    [Show full text]
  • 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.
    [Show full text]
  • Using Peptide-Phage Display to Capture Conditional Motif-Based Interactions
    Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1716 Using peptide-phage display to capture conditional motif-based interactions GUSTAV SUNDELL ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-0433-5 UPPSALA urn:nbn:se:uu:diva-359434 2018 Dissertation presented at Uppsala University to be publicly examined in B42, BMC, Husargatan 3, Uppsala, Friday, 19 October 2018 at 09:15 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Doctor Attila Reményi (nstitute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary). Abstract Sundell, G. 2018. Using peptide-phage display to capture conditional motif-based interactions. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1716. 87 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0433-5. This thesis explores the world of conditional protein-protein interactions using combinatorial peptide-phage display and proteomic peptide-phage display (ProP-PD). Large parts of proteins in the human proteome do not fold in to well-defined structures instead they are intrinsically disordered. The disordered parts are enriched in linear binding-motifs that participate in protein-protein interaction. These motifs are 3-12 residue long stretches of proteins where post-translational modifications, like protein phosphorylation, can occur changing the binding preference of the motif. Allosteric changes in a protein or domain due to phosphorylation or binding to second messenger molecules like Ca2+ can also lead conditional interactions. Finding phosphorylation regulated motif-based interactions on a proteome-wide scale has been a challenge for the scientific community.
    [Show full text]
  • The Immunoassay Guide to Successful Mass Spectrometry
    The Immunoassay Guide to Successful Mass Spectrometry Orr Sharpe Robinson Lab SUMS User Meeting October 29, 2013 What is it ? Hey! Look at that! Something is reacting in here! I just wish I knew what it is! anti-phospho-Tyrosine Maybe we should mass spec it! Coffey GP et.al. 2009 JCS 22(3137-44) True or false 1. A big western blot band means I have a LOT of protein 2. One band = 1 protein Big band on Western blot Bands are affected mainly by: Antibody affinity to the antigen Number of available epitopes Remember: After the Ag-Ab interaction, you are amplifying the signal by using an enzyme linked to a secondary antibody. How many proteins are in a band? Human genome: 20,000 genes=100,000 proteins There are about 5000 different proteins, not including PTMs, in a given cell at a single time point. Huge dynamic range 2D-PAGE: about 1000 spots are visible. 1D-PAGE: about 60 -100 bands are visible - So, how many proteins are in my band? Separation is the key! Can you IP your protein of interest? Can you find other way to help with the separation? -Organelle enrichment -PTMs enrichment -Size enrichment Have you optimized your running conditions? Choose the right gel and the right running conditions! Immunoprecipitation, in theory Step 1: Create a complex between a desired protein (Antigen) and an Antibody Step 2: Pull down the complex and remove the unbound proteins Step 3: Elute your antigen and analyze Immunoprecipitation, in real life Flow through Wash Elution M 170kDa 130kDa 100kDa 70kDa 55kDa 40kDa 35kDa 25kDa Lung tissue lysate, IP with patient sera , Coomassie stain Rabinovitch and Robinson labs, unpublished data Optimizing immunoprecipitation You need: A good antibody that can IP The right beads: i.
    [Show full text]
  • Development of a Phage Display Library for Discovery of Antigenic Brucella Peptides Jeffrey Williams Iowa State University
    Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2018 Development of a phage display library for discovery of antigenic Brucella peptides Jeffrey Williams Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Microbiology Commons Recommended Citation Williams, Jeffrey, "Development of a phage display library for discovery of antigenic Brucella peptides" (2018). Graduate Theses and Dissertations. 16896. https://lib.dr.iastate.edu/etd/16896 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Development of a phage display library for discovery of antigenic Brucella peptides by Jeffrey Williams A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Microbiology Program of Study Committee: Bryan H. Bellaire, Major Professor Steven Olsen Steven Carlson The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this thesis. The Graduate College will ensure this thesis is globally accessible and will not permit alterations after a degree is conferred. Iowa State University
    [Show full text]
  • Computer-Aided Drug Design: a Practical Guide
    Computer-Aided Drug Design: A Practical Guide Forrest Smith, Ph.D. Department of Drug Discovery and Development Harrison School of Pharmacy Auburn, University History of Drug Design • Natural Products-Ebers Papyrus, 1500 B.C. documents over 700 plant based products used to treat a variety of illnesses • The rise of Organic Chemistry, middle or the 20th Century, semi-synthetic and synthetic drugs • Computers emerge in the late 1980s, CADD with minimal impact • Automation in the 1990s, High Through-put Screening and Robotics, Compound Libraries • CADD has continued to advance since its introduction with improving capabilities Computational Chemistry • Ab initio calculations • Semi-empirical calculations • Molecular Mechanics Molecular Mechanics Global Minimum Force Fields • MM2, MM3, MM4 • MMFF • AMBER • CHARM • OPLS Finding the Global Minimum • Systematic Search • Monte Carlo Methods • Simulated Annealing • Quenched Dynamics Minor Groove Binders Computer-Aided Drug Design • Structure Based Design – Docking – Molecular Dynamics – Free Energy Perturbation • Ligand Based Design-QSAR – COMFA – Pharmacophore Modeling – Shape Based Methods Docking-The Receptor • X-Ray Crystal Structures – RCSB Protein Data Bank (https://www.rcsb.org/) – Private Data • Homology Modeling • Nuclear Magnetic Resonance Docking- The Ligand • Proprietary Ligands • Databases qReal Databases • FDA approved drugs- (http://chemoinfo.ipmc.cnrs.fr/MOLDB/index.html) • Purchasable compounds Zinc 15, currently 100 million compounds (http://zinc15.docking.org/) q Virtual Databases-
    [Show full text]
  • Identification of Five Upregulated Genes in Transplanted
    Proc. Nati. Acad. Sci. USA Vol. 91, pp. 6463-6467, July 1994 Medical Sciences Chronic cardiac rejection: Identification of five upregulated genes in transplanted hearts by differential mRNA display (gene expreson/traplant arterlosclerosl/cardlac tnsplatatlon/polymerase chain reaction) ULRIKE UTANS*, PENG LIANG*, LAURI R. WYNERt, MoRRis J. KARNOVSKYt, AND MARY E. RUSSELL*tt§ *Cardiovascular Biology Laboratory, Harvard School of Public Health, tHarvard Medical School, and tCardiovascular Division, Brigham and Women's Hospital, Boston, MA 02115 Communicated by Arthur B. Pardee, March 25, 1994 (receivedfor review November 18, 1993) ABSTRACT Tran t arteriosclerosis, the major man- planted heart that limits transplant survival (3, 4). Studies of festation of chronic rejection, develops after alogeneic (Lewis the process in humans have been restricted by the limited to F344) but not syngeneic (Lewis to Lewis) rat cardiac availability of tissue for analysis. Clinical specimens are transplantation. To identify transcriptionaly regulated medi- heterogeneous in their degree of chronic rejection, their ators asiated with chronic cardiac rejection, we adapted the extent of superimposed disease processes, and the period differential mRNA display technique for in vvo a nt between the time they are obtained and the time of trans- specimens. Gene tanscript patterns In four allogenec hearts plantation. Also, transplanted hearts obtained at autopsy are showing early signs of chronic rejection were compared with not suitable for analysis (which requires viable tissue), and those in two syngeneic hearts exposed to the same surgical the utility of endomyocardial biopsy specimens is limited by procedure but histologically normal. Twelve differentially ex- their small size. Moreover, the restricted extent of arterio- pressed cDNA bands were Identied.
    [Show full text]