Supporting Information
|
|
- Justina Scott
- 6 years ago
- Views:
Transcription
1 Supporting Information Copper and zinc ions specifically promote non-amyloid aggregation of the highly stable human γ-d crystallin Liliana Quintanar, 1,* José A. Domínguez-Calva, 1 Eugene Serebryany, 2 Lina Rivillas- Acevedo, 3 Cameron Haase-Pettingell, 2 Carlos Amero, 3 Jonathan A. King, 2,* 1 Departamento de Química, Centro de Investigación y de Estudios Avanzados (Cinvestav), Mexico City, México 2 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA 3 Centro de Investigaciones Químicas, Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, Cuernavaca, México. jaking@mit.edu; lilianaq@cinvestav.mx Keywords: lens crystallins, human gamma-d crystallin, copper, zinc, cataracts
2 Recombinant HγD crystallin expression and purification. Recombinant HγD crystallin was expressed using a pet16b plasmid in BL21-RIL E. coli, and purified by ammonium sulfate precipitation followed by size exclusion chromatography (SEC). Briefly, this involves growing cells at 37 C in super broth to an optical density of 5 at 600 nm. For the production of 15 N and 13 C doubly labeled recombinant protein, the cells were grown in minimal M9 media containing 15 NH 4 Cl (1g/L, Cambridge Isotopes) and 13 C-glucose (2g/L). In all cases, protein expression was induced with 1 mm isopropyl β-d-1- thiogalactopyranoside (ITPG) at 18 C overnight. Harvested cells from a 2 L culture growth were resuspended with 10 mm ammonium acetate buffer ph 7, 50 mm NaCl, to a total volume of 50 ml. Cells were lysed by incubation with lysozyme (1 mg/ml) and DNase (20 ng/ml) for 30 min, in the presence of EDTA-free Complete-mini protease inhibitor cocktail (Roche), followed by sonication cycles (30 s each) in ice. Lysate was centrifuged at 17,000 g for 45 min, and ammonium sulfate was slowly added to the supernatant to reach 30% (w/v), while stirring in ice. The proteins that precipitated at 30 % ammonium sulfate were discarded, and more ammonium sulfate was added to reach 50% (w/v) and cause HγD crystallin precipitation. The precipitate was resuspended in 10 mm ammonium acetate buffer ph 7, with 50 mm NaCl, spun down, and passed through a 0.2 µm filter before loading into a SEC column (Hi-Prep Sephacryl S-100), using a fast protein liquid chromatography (FPLC) instrument (GE Life Sciences). Eluted HγD crystallin fractions were analyzed by SDS-PAGE to verify purity, pooled and stored at 4 C.
3 Thermal stability measurements by CD and fluorescence. Calculation of the fraction of unfolded protein. The fraction of unfolded (F u ) protein at each temperature was calculated according to Equation 1: F u = ( y-y f )/(y u -y) (Eq. 1) Where y is the CD intensity at 218 nm or the ratio of fluorescence intensity at 350 and 325 nm (FI 350/325) at a given temperature; y u is the intensity associated to the fully unfolded protein, and y N is the intensity associated to the native folded protein. Melting temperatures (T m ) were calculated by determining the midpoints of the thermal transitions where F u = 0.5, as previously described (Greenfield N. J. Nat. Protoc. 2006, 1, and Flaugh, S. L.; Mills, I. A.; King, J. J. Biol. Chem. 2006, 281, ).
4 Figure S1. Effect of temperature in the metal-induced aggregation of HγD crystallin. Turbidity assays of HγD crystallin (50 µm) in the presence of 3 equiv of Cu(II) (A), 10 equiv of Cu(II) (B), or 10 equiv of Zn(II) (C), at 27 C (light blue), 32 C (yellow), 37 C (orange), or 42 C (red). In all cases, the absorbance at 405 nm is reported as function of time after the addition of the metal ion.
5 Figure S2. Initial burst in turbidity is associated to protein aggregation. A rapid increase in turbidity is observed at early times when 10 equiv of Cu(II) are added to HγD crystallin (50 µm) at 37 C (blue trace in A); in contrast, no turbidity is observed when acetate buffer is incubated with 10 equiv of Cu(II) (black trace in A); in both cases, the relative absorbance at 405 nm is reported as function of time. Thus, initial increase in absorption intensity at 405 nm is not associated to Cu absorption, but to formation of protein aggregates. Consistently, the full absorption spectrum collected on the HγD crystallin (50 µm) sample 7 min after the addition of 10 equiv of Cu(II) shows increased turbidity at all wavelengths (blue trace in B). Upon centrifugation of the sample, the spectrum shows no turbidity, and the absorption at 280 nm indicates that only 30% of the protein remains soluble.
6 Figure S3. Thioflavin T assay for the HγD crystallin aggregates. ThT (50 µm) was added to the protein aggregates at the end point of the turbidity assay at 37 C for: HγD crystallin (50 µm) in the absence (grey) and presence of 2, 4, and 10 equiv of Cu(II) (blue traces), or 4 equiv of Zn(II) (red). The emission fluorescence spectrum was collected in each case, using an excitation wavelength of 450 nm, in a Cary Eclipse fluorimeter, and with a 1 cm path length quartz cell. ThT is a dye that fluoresces upon binding to amyloid fibrils. As a positive control, ThT (20 µm) was added to beta-amyloid fibrils that were formed by incubation of beta-amyloid(1-40) peptide (20 µm) in the absence (light green) or presence (dark green) of 1 equiv. of Cu(II) ions at 37 C; the amyloid nature of the beta-amyloid aggregates is revealed by the intense ThT fluorescence with a maximum intensity at 485 nm. In contrast, metal-induced aggregates of HγD crystallin do not bind ThT, and thus, do not cause significant ThT fluorescence; only a small baseline shift is observed at higher concentrations of copper (4 and 10 equiv), due to light scattering by the aggregates. Thus, the nature of the metal-induced aggregates of HγD crystallin is not amyloid.
7 Figure S4. Effect of oxygen in the copper-induced aggregation of HγD crystallin. A) Turbidity assays of HγD crystallin with 4 equiv of Cu(II) in the presence of oxygen (solid line) and under anaerobic conditions (dotted line). B) Aggregates obtained at the end of turbidity assays of HγD crystallin with 0 and 4 equiv of Cu(II) under normal atmospheric environment, and with 4 equiv of Cu(II) under anaerobic conditions.
8 Figure S5. Effect of Cu(II) ions in the folding of HγD crystallin by CD at 37 C. Titration of HγD crystallin (grey spectrum) with 1, 2, 3, 4, 5, 6, 8, and 10 equiv of Cu(II) (light to dark blue traces) was performed at a lower protein concentration (0.5 µm), to allow observation of the positive CD signal at 195 nm, that together with the 218 nm signal, is characteristic of β-sheet folding. Relative CD intensities at 218 and 195 nm are plotted as a function of Cu(II) added in the inset, showing a similar trend.
9 Figure S6. No aggregation occurs in the course of the CD folding experiment (Figure 4A). Titration of HγD crystallin (2 µm grey spectrum) with 1, 2, 3, 4, 5, 6, 8, and 10 equiv of Cu(II) (light to dark blue traces) was performed at 37 C, and followed by CD (A). Simultaneously, the voltage at the CD detector, which is proportional to the absorption of light, was monitored (B), showing no significant changes during the course of the experiment. If the protein were aggregating in the course of the experiment, the scattering of light would be evident in the CD detector voltage. Moreover, a UV-Vis absorption spectrum of the protein solution was collected at the beginning and at the end of the CD experiment, finding practically identical spectra (C). The absorbance at 280 nm is almost the same at the beginning and the end of the experiment, indicating that we are not losing soluble protein during the course of this experiment. Initial absorption at 280 nm corresponds to an initial protein concentration of 2.00 µm, while the final concentration was 1.98 µm.
10 Figure S7. Thermal denaturation curves for HγD crystallin in the presence of Cu(II) and Zn(II), followed by Trp fluorescence. Thermal denaturation of HγD crystallin (2 µm) was done in the presence of 0, 1, 2, 3, 4, or 5 equiv of Cu(II) (A) or Zn(II) (B). In all cases, the fraction of unfolded protein is plotted as a function of temperature. Data for at least duplicate experiments are shown, and the midpoints of the denaturation curves for each condition are listed in Table S1.
11 Table S1. Tm values for HγD crystallin in the presence of Cu(II) or Zn(II) ions. # equiv of Cu(II) Tm ( o C) Tm ( o C) by Fluorescence by CD ± ± ± ± ± ± ± ± ± ± ± ± 2.85 # equiv of Zn(II) Tm ( o C) by CD Tm ( o C) by Fluorescence ± ± ± ± ± ± ± ± ± ±2.41
12 Figure S8. 1H 15N-HSQC spectra of HγD crystallin 100 µm in the absence (black) or presence of 1.5 equivalents of Zn(II) (red) (A), and 1.5 equivalents of Cu(II) (B). The blue square correspond to the amplified region showed in figure 5.
13 Figure S9. 1 H 15 N-HSQC spectra of HγD crystallin 200 µm in the absence (black) or presence of 1.0 equivalent of Mn(II) (red) (A) and 1.0 equivalent of Ca(II) (red) (B). It should be noted that, due to technical issues (broken cryo-probe), this experiment had to be done at a protein concentration that is double than the one used in other NMR experiments, and the signals are broader than in previous experiments. Nevertheless, the signals associated to monomeric protein could be assigned, and they do not change upon addition of Ca(II) or Mn(II).
AFFINITY HIS-TAG PURIFICATION
DESCRIPTION Resins are products that allow batch or column purifications. This product is supplied as a suspension in 50% aqueous suspension containing 30 vol % ethanol. INSTRUCTIONS The resins are adapted
More informationAFFINITY HIS-TAG PURIFICATION
DESCRIPTION Nickel NTA Agarose Cartridges 5ml are used for purification of histidine-tagged proteins in native or denaturing conditions. This cartridge can be used with an automated chromatography system,
More informationNickel-NTA Agarose Suspension
Nickel-NTA Agarose Suspension Agarose beads for purification of His-tagged proteins Product No. A9735 Description Nickel-NTA Agarose Suspension is an agarose-based affinity chromatography resin allowing
More informationINSTRUCTIONS The resins are adapted to work mainly in native conditions like denaturing.
1 AFFINITY HIS-TAG PURIFICATION PROCEDURE FOR USE Nickel NTA Agarose Beads DESCRIPTION Resins are products that allow batch or column purifications. This product is supplied as a suspension in 50% aqueous
More informationPROCEDURE FOR USE NICKEL NTA Magnetic Agarose Beads (5%)
1 AFFINITY HIS-TAG PURIFICATION PROCEDURE FOR USE NICKEL NTA Magnetic Agarose Beads (5%) DESCRIPTION Nickel NTA Magnetic Agarose Beads are products that allow rapid and easy small-scale purification of
More informationAFFINITY HIS-TAG PURIFICATION
DESCRIPTION Resins are products that allow batch or column purifications. This product is supplied as a suspension in 50% aqueous suspension containing 30 vol % ethanol. INSTRUCTIONS The resins are adapted
More informationNi-NTA Agarose. User Manual. 320 Harbor Way South San Francisco, CA Phone: 1 (888) MCLAB-88 Fax: 1 (650)
Ni-NTA Agarose User Manual 320 Harbor Way South San Francisco, CA 94080 Phone: 1 (888) MCLAB-88 Fax: 1 (650) 871-8796 www. Contents Introduction -----------------------------------------------------------------------
More informationCharacterization of mab aggregation using a Cary 60 UV-Vis Spectrophotometer and the Agilent 1260 Infinity LC system
Characterization of mab aggregation using a Cary 60 UV-Vis Spectrophotometer and the Agilent 1260 Infinity LC system Application Note Biopharmaceuticals Authors Arunkumar Padmanaban and Sreelakshmy Menon
More informationINSECT CELL/BACULOVIRUS PRODUCTION
INSECT CELL/BACULOVIRUS PRODUCTION PEF # GENE NAME TRANSFER VECTOR BEVS MOLECULAR WEIGHT 2015-XXXX XXXX pbac1 flashbacultra TM 36.0 kda EXPRESSION METHOD OVERVIEW: Insect cells Spodoptera frugiperda (Sf9)
More information1 ml gel corresponds to ml of 75% (v/v) Glutathione Agarose suspension.
1 AFFINITY GST PURIFICATION Procedure for Use Glutathione Agarose 4 Resin DESCRIPTION Glutathione Agarose Resin is used to purify recombinant derivatives of glutathione S-transferases or glutathione binding
More informationStabilization of a virus-like particle and its application as a nanoreactor at physiological conditions
Supporting Information Stabilization of a virus-like particle and its application as a nanoreactor at physiological conditions Lise Schoonen, b Sjors Maassen, b Roeland J. M. Nolte b and Jan C. M. van
More informationOPPF-UK Standard Protocols: Mammalian Expression
OPPF-UK Standard Protocols: Mammalian Expression Joanne Nettleship joanne@strubi.ox.ac.uk Table of Contents 1. Materials... 3 2. Cell Maintenance... 4 3. 24-Well Transient Expression Screen... 5 4. DNA
More informationSupplemental Information. OprG Harnesses the Dynamics of its Extracellular. Loops to Transport Small Amino Acids across
Structure, Volume 23 Supplemental Information OprG Harnesses the Dynamics of its Extracellular Loops to Transport Small Amino Acids across the Outer Membrane of Pseudomonas aeruginosa Iga Kucharska, Patrick
More informationBIOC 463A Protein Purification Concepts General Concepts about Protein Purification
General Concepts about Protein Purification Initial Considerations: Why do you want the protein (ie. what is your project all about)? How much protein do you need (ng, ug, mg, g)? How homogenous or pure?
More informationThe preparation of native chromatin from cultured human cells.
Native chromatin immunoprecipitation protocol The preparation of native chromatin from cultured human cells. All solutions need to be ice cold. Sucrose containing solutions must be made up fresh on the
More informationTECHNICAL BULLETIN. In Vitro Bacterial Split Fluorescent Protein Fold n Glow Solubility Assay Kits
In Vitro Bacterial Split Fluorescent Protein Fold n Glow Solubility Assay Kits Catalog Numbers APPA001 In Vitro Bacterial Split GFP "Fold 'n' Glow" Solubility Assay Kit (Green) APPA008 In Vitro Bacterial
More informationnanodsf 2bind: Your service provider for biophysical characterization of proteins Precisely revealing protein folding and stability
nanodsf Precisely revealing protein folding and stability 2bind: Your service provider for biophysical characterization of proteins This booklet was written and designed by 2bind 08 2015 Any reproduction
More informationDynamic High Capacity Mustang Q Membrane Units for Scaleable Anion Exchange Chromatography Purification of Adenoviral Vectors
Contact Us: www.pall.com/contact Dynamic High Capacity Mustang Q Membrane Units for Scaleable Anion Exchange Chromatography Purification of Adenoviral Vectors Dynamic High Capacity Mustang Q Membrane Units
More informationPurification of DNA from living cells
Purification of DNA from living cells Total cell DNA & Plasmid DNA Grow and harvest bacterial culture Prepare cell extract Purify DNA from a cell extract Concentrate DNA samples Measure DNA concentration
More informationSUPPLEMENTARY MATERIAL
SUPPLEMENTARY MATERIAL Materials and Methods Circular dichroism (CD) spectroscopy. Far ultraviolet (UV) CD spectra of apo- and holo- CaM and the CaM mutants were recorded on a Jasco J-715 spectropolarimeter
More informationAn effective platform for purification of IgM monoclonal antibodies using Hydroxyapatite
An effective platform for purification of IgM monoclonal antibodies using Hydroxyapatite Frank Hensel, Patrys, GmbH Pete Gagnon, Validated Biosystems 5th International Conference on Hydroxyapatite and
More informationSensoLyte Anti-alpha-Synuclein Quantitative ELISA Kit (Human/Mouse/Rat) *Colorimetric*
Catalog # Kit Size SensoLyte Anti-alpha-Synuclein Quantitative ELISA Kit (Human/Mouse/Rat) *Colorimetric* AS-55550 One 96-well strip plate This kit is optimized to detect human/mouse/rat alpha-synuclein
More informationRNAprotect Bacteria Reagent Handbook
January 2015 RNAprotect Bacteria Reagent Handbook RNAprotect Bacteria Reagent For in vivo stabilization of total RNA in bacteria RNeasy Protect Bacteria Mini Kit RNeasy Protect Bacteria Midi Kit For in
More informationO-GlcNAcase Activity Assay
O-GlcNAcase Activity Assay Prepared by Jen Groves and Junfeng Ma, The Johns Hopkins Unviersity School of Medicine Based on: Macauley MS et al., 2005. O-GlcNAcase uses substrate-assisted catalysis: kinetic
More informationProtein Expression Research Group (PERG) 2012 Study
Protein Expression Research Group (PERG) 2012 Study Richard J Heath 1, Fei P Gao 2, Pamela Scott Adams 3, Cynthia Kinsland 4, James Bryson 5, Bo Xu 6, Thomas Neubert 7. 1 St Jude Children's Research Hospital,
More informationStrep-Tactin XT Spin Column
Strep-Tactin XT Spin Column Purification Protocol Last date of revision Last June date 2017 of revision June 2017 Version PR90-0001 Version PR90-0001 For research use only Important licensing information
More informationPurification: Step 1. Lecture 11 Protein and Peptide Chemistry. Cells: Break them open! Crude Extract
Purification: Step 1 Lecture 11 Protein and Peptide Chemistry Cells: Break them open! Crude Extract Total contents of cell Margaret A. Daugherty Fall 2003 Big Problem: Crude extract is not the natural
More informationPurification: Step 1. Protein and Peptide Chemistry. Lecture 11. Big Problem: Crude extract is not the natural environment. Cells: Break them open!
Lecture 11 Protein and Peptide Chemistry Margaret A. Daugherty Fall 2003 Purification: Step 1 Cells: Break them open! Crude Extract Total contents of cell Big Problem: Crude extract is not the natural
More informationE.Z.N.A. Blood DNA Midi Kit. D preps D preps
E.Z.N.A. Blood DNA Midi Kit D3494-00 2 preps D3494-04 100 preps August 2013 E.Z.N.A. Blood DNA Midi Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Preparing
More informationBefore You Begin. Calibration Protocols
Before You Begin Read through this entire protocol sheet carefully before you start your experiment and prepare any materials you may need. Calibration Protocols 1. OD 600 Reference point You will use
More informationStrep-Tactin Spin Column Purification Protocol
Strep-Tactin Spin Column Purification Protocol Last date of revision February 2008 Version PR10-0005 IBA Headquarters IBA GmbH Rudolf-Wissell-Str. 28 D-37079 Göttingen Germany Tel: +49 (0) 551-50672-0
More informationMethod for Folding of Recombinant Prion Protein to Soluble β-sheet Secondary Structure
Chapter 2 Method for Folding of Recombinant Prion Protein to Soluble β-sheet Secondary Structure Laura J. Ellett Abstract A key event in the pathogenesis of prion diseases is the change in structure of
More informationProtein Stability Analysis Using the Optim Patrick Celie NKI Protein Facility, Amsterdam
Protein Stability Analysis Using the Optim 1000 Patrick Celie NKI Protein Facility, Amsterdam NKI Protein Facility Fundamental and translation cancer research ~ 650 scientists + supporting personnel Connected
More informationPurification of mfp. from an Overnight Culture. Laboratory 17
Purification of mfp from an Overnight Culture When scientists at a therapeutics company, like Amgen, have successfully identified a promising therapeutic protein, two objectives would be to locate and
More informationCase 7 A Storage Protein From Seeds of Brassica nigra is a Serine Protease Inhibitor Last modified 29 September 2005
Case 7 A Storage Protein From Seeds of Brassica nigra is a Serine Protease Inhibitor Last modified 9 September 005 Focus concept Purification of a novel seed storage protein allows sequence analysis and
More informationBIOC 463A Protein Purification Concepts General Concepts about Protein Purification
General Concepts about Protein Purification Initial Considerations: Why do you want the protein (ie. what is your project all about)? How much protein do you need (ng, ug, mg, g)? How homogenous or pure?
More informationab GFP ELISA Kit Instructions for Use For the quantitative measurement of GFP protein expression
ab117992 GFP ELISA Kit Instructions for Use For the quantitative measurement of GFP protein expression This product is for research use only and is not for diagnostic use. intended www.abcam.com Table
More informationab65354 Superoxide Dismutase Activity Assay kit (Colorimetric)
Version 9 Last updated 11 January 2018 ab65354 Superoxide Dismutase Activity Assay kit (Colorimetric) For the measurement of Superoxide Dismutase Activity in various samples. This product is for research
More informationThe practical task of monoclonal IgG purification with CHT TM ceramic hydroxyapatite
The practical task of monoclonal IgG purification with CHT TM ceramic hydroxyapatite Pete Gagnon, Jie He, Paul Ng, Julia Zhen, Cheryl Aberin, Heather Mekosh 11th Annual Waterside Conference, Chicago, May
More informationN-terminal dual protein functionalization by strainpromoted alkyne nitrone cycloaddition
Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry -terminal dual protein functionalization by strainpromoted alkyne nitrone cycloaddition Rinske P. Temming, a Loek Eggermont,
More informationARBRE-P4EU Consensus Protein Quality Guidelines for Biophysical and Biochemical Studies Minimal information to provide
ARBRE-P4EU Consensus Protein Quality Guidelines for Biophysical and Biochemical Studies Minimal information to provide Protein name and full primary structure, by providing a NCBI (or UniProt) accession
More informationAmintra Affinity Resins
Amintra Affinity Resins Ni-NTA Metal Chelate Affinity Resin Technical Data and Instruction Manual info@expedeon.com 2 TABLE OF CONTENTS TABLE OF CONTENTS 3 Introduction 4 Storage 4 Chemical compatibility
More informationThe Production of a Recombinant Biotechnology Product. Chapter 8
The Production of a Recombinant Biotechnology Product Chapter 8 Objectives Give a basic overview of genetic engineering. Describe the processes involved in isolating a piece DNA of interest Mass producing
More informationA General Protocol for GST Pull-down Lili Jing *
A General Protocol for GST Pull-down Lili Jing * Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA *For correspondence: lilijingcn@gmail.com [Abstract] GST pull-down
More informationPurification of (recombinant) proteins. Pekka Lappalainen, Institute of Biotechnology, University of Helsinki
Purification of (recombinant) proteins Pekka Lappalainen, Institute of Biotechnology, University of Helsinki Physical properties of proteins that can be applied for purification -size -charge (isoelectric
More informationAurum Plasmid Mini Kit. Instruction Manual. Bio-Rad Laboratories, Inc Alfred Nobel Dr. Hercules, CA USA (510)
Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr. Hercules, CA 94547 USA (510) 741-1000 1-800-424-6723 Aurum Plasmid Mini Kit Instruction Manual For technical service, call your local Bio-Rad office, or
More informationE.Z.N.A. Water DNA Kit. D preps D preps D preps
E.Z.N.A. Water DNA Kit D5525-00 5 preps D5525-01 50 preps D5525-02 200 preps April 2017 E.Z.N.A. Water DNA Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Preparing
More informationE.Z.N.A. MicroElute Genomic DNA Kit. D preps D preps D preps
E.Z.N.A. MicroElute Genomic DNA Kit D3096-00 5 preps D3096-01 50 preps D3096-02 200 preps December 2013 E.Z.N.A. MicroElute Genomic DNA Kit Table of Contents Introduction...2 Kit Contents/Storage and Stability...3
More informationE.Z.N.A. Yeast Plasmid Mini Kit. D preps D preps
E.Z.N.A. Yeast Plasmid Mini Kit D3376-00 5 preps D3376-01 50 preps November 2015 E.Z.N.A. Yeast Plasmid Mini Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Preparing
More information1. Bloomsbury BBSRC Centre for Structural Biology, Birkbeck College and University College London.
Purification/Polishing of His-tagged proteins - Application of Centrifugal Vivapure Ion-exchange Membrane Devices to the Purification/Polishing of Histagged Background Multi-milligram quantities of highly
More informationBio-Scale Mini Profinity IMAC Cartridges, 1 and 5 ml. Instruction Manual. Catalog #
Bio-Scale Mini Profinity IMAC Cartridges, 1 and 5 ml Instruction Manual Catalog # 732-4610 732-4612 732-4614 Table of Contents Section 1...Introduction...1 Section 2 Product Information...2 Section 3 Connection
More informationSUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION Conserved arginines on the rim of Hfq catalyze base pair formation and exchange Subrata Panja and Sarah A. Woodson T.C. Jenkins Department of Biophysics, Johns Hopkins University,
More informationProduct # (10 preps) Lysis Additive A Binding Buffer I. Elution Buffer B
3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com Soil DNA Isolation Maxi Kit Product # 62000 Product Insert Norgen
More informationPROTOCOL 1: EXTRACTION OF DNA FROM BACTERIA
PROTOCOL 1: EXTRACTION OF DNA FROM BACTERIA The basic standard procedures for isolation of bacterial DNA are based on lysozyme digestion of the cell wall, detergent lysis, disruption of protein-nucleic
More informationSupporting Information
Supporting Information Wiley-VCH 26 69451 Weinheim, Germany A new homogenous assay for studying mira maturation Brian Patrick Davies and Christoph Arenz General Information For MALDI-TF measurements a
More informationPurification of His-tag proteins
Purification of His-tag proteins User manual Protino Ni-TED 150 Packed Columns Protino Ni-TED 1000 Packed Columns Protino Ni-TED 2000 Packed Columns Protino Ni-TED Resin July 2017 / Rev. 07 www.mn-net.com
More informationCell Growth and DNA Extraction- Technion igem HS
Growing Cells and DNA Extraction Goals 1. Become familiar with the process of growing bacteria 2. Get to know the DNA extraction process 3. Perform miniprep in the lab Keywords 1. Growth stages 6. Techniques
More informationBio-Scale Mini Nuvia IMAC Ni-Charged Cartridges, 1 and 5 ml
Bio-Scale Mini Nuvia IMAC Ni-Charged Cartridges, 1 and 5 ml Instruction Manual Catalog numbers 780-0811 780-0812 Table of Contents Section 1 Introduction... 1 Section 2 Product Information... 2 Section
More informationEZ-10 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK
EZ-0 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK (Bacteria, Plant, Animal, Blood) Version 8 Rev 05/0/03 EZ-0 Genomic DNA Kit Handbook Table of Contents Introduction Limitations of Use Features Applications
More informationVectors for Gene Cloning: Plasmids and Bacteriophages
Vectors for Gene Cloning: Plasmids and Bacteriophages DNA molecule must be able to replicate within the host cell to be able to act as a vector for gene cloning, so that numerous copies of the recombinant
More informationAnaTag HiLyte Fluor 647 Protein Labeling Kit
AnaTag HiLyte Fluor 647 Protein Labeling Kit Catalog # 72049 Kit Size 3 Conjugation Reactions This kit is optimized to conjugate HiLyte Fluor 647 SE to proteins (e.g., IgG). It provides ample materials
More informationSupporting Online Material, Matsumoto et al.
Supporting Online Material, Matsumoto et al. Material and Methods Library. Poly(A) + mrna was purified from RAW264.7 cells stimulated with murine IFN-γ (100 units/ml) and bacterial LPS (100 ng/ml) for
More information1. Cross-linking and cell harvesting
ChIP is a powerful tool that allows the specific matching of proteins or histone modifications to regions of the genome. Chromatin is isolated and antibodies to the antigen of interest are used to determine
More informationFor Research Use Only Ver
INSTRUCTION MANUAL ZR-Duet DNA/RNA MiniPrep Plus Catalog No. D7003 Highlights Efficient isolation and separation of DNA and RNA from any cells, tissue, blood, and biological fluids. High quality DNA and
More informationab83355 ATP Assay Kit (Colorimetric/ Fluorometric)
Version 18 Last updated 2 January 2018 ab83355 ATP Assay Kit (Colorimetric/ Fluorometric) For the rapid, sensitive and accurate measurement of ATP in a variety of samples. This product is for research
More informationE.Z.N.A. Blood DNA Maxi Kit. D preps D preps
E.Z.N.A. Blood DNA Maxi Kit D2492-00 2 preps D2492-03 50 preps April 2014 E.Z.N.A. Blood DNA Maxi Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Preparing Reagents...4
More informationE.Z.N.A. Bacterial RNA Kit. R preps R preps
E.Z.N.A. Bacterial RNA Kit R6950-00 5 preps R6950-01 50 preps July 2017 E.Z.N.A. Bacterial RNA Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Before Beginning...4
More informationStrep-Tactin Spin Column
Strep-Tactin Spin Column Purification Protocol Last date of revision November 2012 Version PR10-0006 www.strep-tag.com For research use only Important licensing information Products featuring Strep-Tactin
More informationLow cost and non-toxic genomic DNA extraction for use in molecular marker studies.
Low cost and non-toxic genomic DNA extraction for use in molecular marker studies. Version 1.4, February 28 th, 2013. Prepared by Bernhard Hofinger, Owen Huynh and Brad Till. 1. OBJECTIVE To develop and
More informationImaging of protein crystals with two photon microscopy
Supporting Information Imaging of protein crystals with two photon microscopy Pius Padayatti,*, Grazyna Palczewska,*, Wenyu Sun, Krzysztof Palczewski,# and David Salom Polgenix Inc., Cleveland, Ohio 44106,
More informationInstructions for Use Life Science Kits & Assays
Instructions for Use Life Science Kits & Assays Content Content 1 Product and order number... I 2 Storage conditions... I 3 Description... II 3.1 Quality data... II 3.2 Unit definition... II 4 Delivered
More informationSYSTEMATIC STUDY OF THE DIFFERENT QUENCHING PHENOMENA IN ORGANIC SCINTILLATORS
SYSTEMATIC STUDY OF THE DIFFERENT QUENCHING PHENOMENA IN ORGANIC SCINTILLATORS Luz Santiago, Hector Bagán, Alex Tarancón Sanz, Gemma Rauret, Jose Francisco García Departmento de Química Analitica de la
More informationCellular Fractionation
Cellular Fractionation Lamond Lab Protocol 2007 More detailed protocol can be found here: http://www.lamondlab.com/f7nucleolarprotocol.htm This protocol has been adapted to fractionate a variety of different
More informationPrimeScript RT Master Mix (Perfect Real Time)
Cat. # RR036A For Research Use PrimeScript RT Master Mix (Perfect Real Time) Product Manual Table of Contents I. Description... 3 II. Kit Components... 3 III. Materials Required but not Provided... 3 IV.
More informationE.Z.N.A. Stool DNA Kit. D preps D preps D preps
E.Z.N.A. Stool DNA Kit D4015-00 5 preps D4015-01 50 preps D4015-02 200 preps April 2013 E.Z.N.A. Stool DNA Kit Table of Contents Introduction and Overview...2 Illustrated Protocol...3 Kit Contents/Storage
More informationLecture 8: Affinity Chromatography-III
Lecture 8: Affinity Chromatography-III Key words: Chromatography; Affinity chromatography; Protein Purification During this lecture, we shall be studying few more examples of affinity chromatography. The
More informationIntroduction to Protein Purification
Introduction to Protein Purification 1 Day 1) Introduction to Protein Purification. Input for Purification Protocol Development - Guidelines for Protein Purification Day 2) Sample Preparation before Chromatography
More informationActive targeting of the nucleus using non-peptidic boronate tags
Supporting Information Active targeting of the nucleus using non-peptidic boronate tags Rui Tang 1, Ming Wang 2, Moumita Ray 1, Ying Jiang 1, Ziwen Jiang 1, Qiaobing Xu 2 *, Vincent M. Rotello 1 * 1 Department
More informationSpecifications. Kit Specifications. Alcohol Precipitation: Up to 100 ml Column Purification: Up to 5 ml Column Binding Capacity 25 µg
3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com BAC DNA MiniPrep Kit Product # 18050 Product Insert The BAC DNA
More informationNoviPure Microbial Protein Kit (50)
Saving You Time For Life NoviPure Microbial Protein Kit (50) Catalog No. 47044 Quantity: 50 preps INSTRUCTION MANUAL Version 04202016 Please recycle www.mobio.com T: 800-606-6246 T: 760-929-9911 technical@mobio.com
More informationAffinity Chromatography. Teaching Kit Manual. GeNei TM. Cat No. New Cat No. KT Revision No.:
Affinity Chromatography Teaching Kit Manual Cat No. New Cat No. KT41 106192 Revision No.: 00010905 CONTENTS Page No. Objective 3 Principle 3 Kit Description 4 Materials Provided 6 Procedure 7 Result 12
More informationE.Z.N.A. Blood DNA Mini Kit. D preps D preps D preps
E.Z.N.A. Blood DNA Mini Kit D3392-00 5 preps D3392-01 50 preps D3392-02 200 preps January 2017 E.Z.N.A. Blood DNA Mini Kit Table of Contents Introduction and Overview...2 Illustrated Protocol...3 Kit Contents/Storage
More informationViral RNAi suppressor reversibly binds sirna to. outcompete Dicer and RISC via multiple-turnover
Supplementary Data Viral RNAi suppressor reversibly binds sirna to outcompete Dicer and RISC via multiple-turnover Renata A. Rawlings 1,2, Vishalakshi Krishnan 2 and Nils G. Walter 2 * 1 Biophysics and
More informationRenaturation Basic Kit for Proteins
Renaturation Basic Kit for Proteins Product Number 96827 Store at 2-8 C Application The Renaturation Basic Kit for Proteins is a rapid empirical screening method used to determine the best conditions for
More informationA nucleic acid-based fluorescent sensor for expeditious detection of pyrophosphate anions at nanomolar concentrations
Supporting Information for A nucleic acid-based fluorescent sensor for expeditious detection of pyrophosphate anions at nanomolar concentrations Xin Su, Chen Zhang, Xianjin Xiao, Anqin Xu, Zhendong Xu
More informationE.Z.N.A. Stool DNA Kit. D preps D preps D preps
E.Z.N.A. Stool DNA Kit D4015-00 5 preps D4015-01 50 preps D4015-02 200 preps July 2017 E.Z.N.A. Stool DNA Kit Table of Contents Introduction and Overview...2 Illustrated Protocol...3 Kit Contents/Storage
More informationSupporting Information
Supporting Information Wiley-VCH 2006 69451 Weinheim, Germany Rolling-circle Amplification of a DNA Nanojunction Chenxiang Lin, Mingyi Xie, Julian J.L. Chen, Yan Liu and Hao Yan A. RCA replication of the
More informationHiPer Yeast Genomic DNA Extraction Teaching Kit
HiPer Yeast Genomic DNA Extraction Teaching Kit Product Code: HTBM013 Number of experiments that can be performed: 10 Duration of Experiment: 3 days Day 1: Revival of Host Day 2: Inoculation of culture
More informationHiPer Gel Extraction Teaching Kit (Column Based)
HiPer Gel Extraction Teaching Kit (Column Based) Product Code: HTBM010 Number of experiments that can be performed: 10 Duration of Experiment Agarose Gel Electrophoresis: 1 hour Protocol: 1 hour Agarose
More informationSurePrep Soil DNA Isolation Kit
1 Reagent Lane, Fair Lawn, NJ 07410 Phone: (201)-796-7100 Fa x: (201) 703-3159 Email: chem.techinfo@thermofisher.com SurePrep Soil DNA Isolation Kit Product Cat. # BP2815-50 Instruction Manual I. Introduction
More informationE.Z.N.A. mirna Kit. R preps R preps R preps
E.Z.N.A. mirna Kit R7034-00 5 preps R7034-01 50 preps R7034-02 200 preps August 2011 E.Z.N.A. Micro RNA Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Preparing
More informationab GST 6XHis-tag ELISA Kit For the quantitative measurement of 6XHis-tag protein expression
ab128573 GST 6XHis-tag ELISA Kit Instructions for Use For the quantitative measurement of 6XHis-tag protein expression This product is for research use only and is not intended for diagnostic use. 1 Table
More informationAmplicon Library Preparation Method Manual. GS FLX Titanium Series October 2009
GS FLX Titanium Series 1. Workflow 3. Procedure The procedure to prepare Amplicon libraries is shown in Figure 1. It consists of a PCR amplification, performed using special Fusion Primers for the Genome
More informationApplication Note. Author. Abstract. Biopharmaceuticals. Verified for Agilent 1260 Infinity II LC Bio-inert System. Sonja Schneider
Combining small-scale purification and analysis of monoclonal antibodies on one instrument Protein purification with high-volume injection using the Agilent 126 Infinity Bio-inert Quaternary LC System
More informationApplication Note USD Purification of Mouse IgM from Cell Culture Supernatant by Cation Exchange Chromatography on CM Ceramic HyperD F Sorbent
Application Note USD 241 Purification of Mouse IgM from Cell Culture Supernatant by Cation Exchange Chromatography on CM Ceramic HyperD F Sorbent What this Study Demonstrates T h i s s t u d y o n C a
More information64 CuCl 2 in 50 µl 0.1N NaOAc buffer, and 20 µg of each DOTA-antibody conjugate in 40 µl
Number of DOTA per antibody The average number of DOTA chelators per antibody was measured using a reported procedure with modifications (1,2). Briefly, nonradioactive CuCl 2 (80-fold excess of DOTA antibodies)
More informationPURIFICATION, SUBUNIT DETERMINATION,
7/25/2008 UCLA CHEM 153L BIOCHEMICAL METHODS I SUMMER 2008 PROFESSOR STEVEN J. KIM TA MAURICE SECTION 1C GROUP MOO0OO PURIFICATION, SUBUNIT DETERMINATION, AND KINETICS OF LACTATE DEHYDROGENASE REPORT BY
More informationProtein Purification Products. Complete Solutions for All of Your Protein Purification Applications
Protein Purification Products Complete Solutions for All of Your Protein Purification Applications FLAG-Tagged Protein Products EXPRESS with the pcmv-dykddddk Vector Set Fuse your protein of interest to
More informationBio-Monolith Protein G Column - More Options for mab Titer Determination
Bio-Monolith Protein G Column - More Options for mab Titer Determination Application Note Biologics and Biosimilars Author Phu T. Duong Agilent Technologies, Inc. Introduction In recent years, monoclonal
More informationHuman Amyloid Beta Peptide 1-42 (Aβ1-42) ELISA Kit
Human Amyloid Beta Peptide 1-42 (Aβ1-42) ELISA Kit Catalog Number. For the quantitative determination of human amyloid beta peptide 1-42 (Aβ1-42) concentrations in serum, plasma, tissue homogenates, cerebrospinal
More information