Telomere and telomerase (1)

Telomere and telomerase assay

A telomere is a region of repetitive DNA at the end of a chromosome, which protects the end of the chromosome from deterioration. Its name is derived from the Greek nouns telos (τἐλος) "end" and merοs (μέρος, root: μερ-) "part".

Russian theorist Alexei Olovnikov was the first to recognize (1971) the problem of how chromosomes could replicate right to the tip, as such was impossible with replication in a 5' to 3' direction. To solve this and to accommodate Leonard Hayflick's idea of limited somatic cell division, Olovnikov suggested that DNA sequences would be lost in every replicative phase until they reached a critical level, at which point cell division would stop.

During cell division, the enzymes that duplicate the chromosome and its DNA cannot continue their duplication all the way to the end of the chromosome. If cells divided without telomeres, they would lose the ends of their chromosomes, and the necessary information they contain. (In 1972, James Watson named this phenomenon the "end replication problem".) The telomeres are disposable buffers blocking the ends of the chromosomes and are consumed during cell division and replenished by an enzyme, the telomerase reverse transcriptase.

Elizabeth Blackburn compared telomeres to the aglets (tips) on the ends of shoelaces that keep them from fraying.

In 1975–1977, Blackburn, working as a postdoctoral fellow at Yale University with Joseph Gall, discovered the unusual nature of telomeres, with their simple repeated DNA sequences composing chromosome ends. Their work was published in 1978. The telomere shortening mechanism normally limits cells to a fixed number of divisions, and animal studies suggest that this is responsible for aging on the cellular level and sets a limit on lifespans. Telomeres protect a cell's chromosomes from fusing with each other or rearranging—abnormalities which can lead to cancer—and so cells are normally destroyed when their telomeres are consumed. Most cancers are the result of "immortal" cells which have ways of evading this programmed destruction.

Elizabeth Blackburn, Carol Greider, and Jack Szostak were awarded the 2009 Nobel Prize in Physiology or Medicine for the discovery of how chromosomes are protected by telomeres and the enzyme telomerase.

Telomeres and telomerase: biological and clinical importance ...
(15) developed an assay for testing telomerase activity in cell extracts. Based on identification of telomerase mechanisms and properties, the telomere ...
www.clinchem.org/cgi/content/full/43/5/708

Telomere Length Analysis and In Vitro Telomerase Assay.

1 Introduction; 2 Materials and Methods; 2.1 Telomere Measurement; 2.2 Telomere Cloning; 2.3 TRAP Telomerase Assay; 3 Notes; References. Browse by Subject ...
www.springerprotocols.com/Abstract/doi/10.../1-59259-434-4:123

Facts about Telomeres and Telomerase

The following section will teach you the basics of telomeres and telomerase. It will also introduce you to the potential applications of current telomerase ...
www4.utsouthwestern.edu/cellbio/shay.../sw_facts.html

TRAP–LIG, a modified telomere repeat amplification protocol assay to quantitate telomerase inhibition by small molecules.
Its major function is to maintain the length of telomeric DNA by synthesizing telomeric DNA repeats, and its enzymatic activity is assayed by means of the ...
linkinghub.elsevier.com/retrieve/pii/S0003269708002960

Telomeres and Telomerase: The Cellular Timekeepers

The telomeres appear to be the cellular timekeepers, but are they related to human aging?
www.senescence.info/telomeres.html

Effect of Telomere and Telomerase Interactive Agents on Human Tumor and Normal Cell Lines.
The cytotoxic effect of telomere and telomerase interactive agents against normal human marrow cells in the colony-forming assay showed the same range of ...
clincancerres.aacrjournals.org/content/6/3/987.full

Cell Research - Telomere and telomerase in oncology
Shortening of the telomeric DNA at the chromosome ends is presumed to limit the lifespan of... Telomere, telomerase, cancer, telomerase assay, inhibitor ...
www.nature.com

Telomeres and telomerase — Genes & Development
Cdc13p (gray) and Est1 (purple) may recruit telomerase to the telomeric 3′ end in ..... (1995) An in vitro assay for Saccharomyces telomerase requires EST1. ...
genesdev.cshlp.org/content/13/18/2353.full

Telomerase Assay in Renal Cancer

Bacchetti, S. and Counter, C. M. (1995) Telomeres and telomerase in human cancer. ...quantitative nonisotopic assay for telomerase activity in humantumors. ...
www.springerprotocols.com/Abstract/doi/10.1385/1-59259-144-2:05

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1. Expression and Purification of Soluble and Correctly Folded Bovine β-Lactoglobulin Variants A and B in Escherichia coli for NMR Studies
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 16 December 2009
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2. Expression of soluble and functional human neonatal Fc receptor in Pichia pastoris
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 16 December 2009
Chang-Han Lee, Dong-Ki Choi, Hye-Ji Choi, Moo-Young Song, Yong-Sung Kim

3. Formate-nitrate transporters: optimisation of expression, purification and analysis of prokaryotic and eukaryotic representatives
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 16 December 2009
Katherine S.H. Beckham, Jane A. Potter, Shiela E. Unkles

4. Streamlined, automated protocols for the production of milligram quantities of untagged recombinant human cyclophilin-A (hCypA) and untagged human proliferating cell nuclear antigen (hPCNA) using ÄKTAxpress™
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5. Expression, purification and primary crystallographic study of human androgen receptor in complex with DNA and coactivator motifs
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6. Purification and characterization of recombinant CH3 domain fragment of the CREB-binding protein
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7. Establishment of a simple and rapid method to screen for strong promoters in Bacillus subtilis
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9. Establishment of a simple and rapid method to screen for strong promoters in Bacillus subtilis
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Protein Purification (from Wikipedia, the Free Encyclopedia)

Contents

1 Purpose

2 Strategies

3 Evaluating purification yield

4 Purification of a tagged protein

5 Methods of protein purification

6 Extraction

7 Precipitation and differential solubilization

8 Ultracentrifugation

9 Chromatographic methods

9.1 Size exclusion chromatography

9.2 Separation based on charge or hydrophobicity

9.3 Ion exchange chromatography

9.4 Affinity chromatography

9.4.1 Metal binding

9.4.2 Immunoaffinity chromatography

9.5 HPLC

10 Concentration of the purified protein

10.1 Lyophilization

10.2 Ultrafiltration

11 Analytical

11.1 Denaturing-Condition Electrophoresis

11.2 Non-Denaturing-Condition Electrophoresis

12 References

13 External links

Protein Purification and Identification Protocols

Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterisation of the function, structure and interactions of the protein of interest. The starting material is usually a biological tissue or a microbial culture. The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps exploit differences in protein size, physico-chemical properties and binding affinity.

1. ALKYL Aspartamide HIC Columns

2. Comparison of affinity tags for protein purification.

3. DEAE Column Preparation

4. DEAE column: FPLC

5. Green Fluorescent Protein Isolation (Gary J. Lindquester)

6. GST Fusion Protein Preparation

7. GST Fusion Protein Purification from Yeast

8. GST-Fusion Protein Purification

9. HeLa Cell Nuclei Preparation (John Garland, The Biochemistry and Molecular Biology Department of The James H. Quillen College of Medicine in Johnson City, TN)

10. HILIC and SEC Columns PolySULFOETHYL and PolyHYDROXYETHYL Columns

11. Isolation of Protein from Tissue (Reddy research laboratory, Neurological Sciences Institute, Oregon Health & Science University)

12. Making Affinity Columns

13. Making Conductivity Curves (Waters Lab)

14. PI-PLC Purification (Bjorkman Group, Howard Hughes Medical Institute at California Institute of Technology)

15. Polyaspartic Acid Weak Cation Exchange Columns

16. PolyGLYCOPLEX Complex Carbohydrates Columns

17. PolySULFOETHYL Aspartamide Strong Cation Exchange Columns

18. PolyWAX LP Weak Anion Exchange Columns

19. Preparation of Tonsil Lysate (Springer Lab, Harvard University)

20. Protein Extraction (Frank Bottone Jr.)

21. Protein Extraction from Tissues (Reddy research laboratory, Neurological Sciences Institute, Oregon Health & Science University)
Protein Isolation Protocols (Lamond Lab, University of Dundee)

22. Protein Isolation using Trizol (Frank Bottone Jr.)

23. Protein Purification

24. Protein Purification

25. Protein Purification

26. Protein Purification

27. Protein Purification (handbook)

28. Protein Purification (Michael Blaber)

29. Protein Purification (Michael Blaber)

30. Protein Purification (Michael Blaber)

31. protein purification (Pictures)

32. Protein Purification and Analysis Protocols and Applications Guide (Promega)

33. Protein Purification on 1G3 Column (Bjorkman Group, Howard Hughes Medical

34. Protein Purification Protocols (The Protein Expression and Purification Facility, EMBL)

35. Protein Sample Preparation & Protein Purification (Invitrogen)

36. Purificaiton of Protein by Affinity Chromatography (Krause Lab)

37. Purification of GST Fused Proteins

38. Purification of GST Fused Proteins

39. Purification of His-tagged proteins from Sf9 cells under native conditions

40. Purification of Proteins (NCBI)

41. Purification Scheme for Ubiquitin Expression Lysate (Sosnick Lab, University of Chicago)

42. Purification of Maltose Binding Proteins Fused Proteins

43. Purification of MBP (maLTose-Binding Proteins) Fused Proteins (Bowtell Lab

44. Removal of Nucleic Acids from Protein (Dr. Mario Lebendiker, The Hebrew University of Jerusalem)

45. RNA and protein extraction from the same tissue (Ivan J Delgado)

46. SDS Removal Guard Columns

47. Small Scale MBP-fusion Protein Purification (Dr. Mario Lebendiker, The Hebrew University of Jerusalem)

48. Subcellular Fractionation of Proteins (Bowtell Lab Manual, Peter MacCallum Cancer Institute)

49. Sucrose Density Gradient Fractionation of Yeast Membranes (Dohlman Lab)
Test Tube for Ion Exchange Chromatography (Dr. Mario Lebendiker, The Hebrew University of Jerusalem)

50. The Protein Purification Facility (Dr. Mario Lebendiker)

51. Tips for acid elution of peptides (Bjorkman Group, Howard Hughes Medical Institute at California Institute of Technology)

52. Whole Cell Protein Extracts (Morrisey Lab, University of Pennsylvania)
This is for both cytoplasmic and nuclear proteins.
Protein Purification - How to Purify Proteins.

Suggested Reading

Protein Purification HandbookWhat is Biotechnology?Protein Engineering MethodsWhat is a Protein?What are Enzymes?

Databases of Virology

The Journal of Virology Online - http://jvi.asm.org/

Online version of the Journal of Virology, the premier journal for peer-reviewed papers on virology research.

Retrovirology - http://www.retrovirology.com
A peer reviewed journal that publishes reviews and original research on retroviruses.

Virus Databases Online - http://www.ictvdb.rothamsted.ac.uk/
A comprehensive index of viruses provided by the International Committee on Taxonomy of Viruses. Also provides a picture gallery, identification tool and software tools.

All the Virology on the WWW - http://www.virology.net/
Links to virology information, collection of virus pictures, virology bookshop, jobs, and course notes.

UK Clinical Virology Network - http://www.clinical-virology.org
News, clinical tests, and current viruses in circulation.

HIV Sequence Database - http://www.hiv.lanl.gov/content/index
Collection HIV and related genetic sequence data and computer analyses - from the Los Alamos Natl. Lab.

American Society for Virology - http://www.asv.org/
A forum for investigators of human, animal, insect, plant, fungal and bacterial viruses in areas such as clinical, ecological, biological or biochemical research.

Paramyxoviruses - http://en.wikipedia.org/wiki/Paramyxovirus
Information from Wikipedia on these negative-sense single-stranded RNA viruses, responsible for a number of human and animal diseases, including their taxonomy, morphology and pathogenesis.

Poxvirus Bioinformatics Resource - http://www.poxvirus.org
The Poxvirus Bioinformatics Resource Center has been established to provide resources to the scientific community for basic research and to facilitate the development of novel antiviral therapies and vaccines against human orthopoxvirus infections as well as approaches for environmental detection of virions and the rapid diagnosis of disease.

Viral Replication - http://en.wikipedia.org/wiki/Viral_replication
Information from Wikipedia on the attachment, entry, and expression of viral genomic material in host cells.

Wadsworth Center - New York State Department of Health - http://www.wadsworth.org/databank/viruses.htm
Information and images regarding; Rabies virus, Herpes virus, Enteroviruses, Norwalk virus, Human Parvovirus B19 and Rotavirus.

Virology Down Under - http://www.uq.edu.au/vdu/
An Australian based portal and educational site providing links and information on human viruses and related areas of science.

Hidden Killers--Deadly Viruses - http://library.thinkquest.org/23054/gather/index.shtml
Basics about viruses, our bodies' immune systems, information about specific viruses, and how these 'hidden killers' could potentially be used as bio-weapons. There are also weekly/bi-weekly feature articles on various facets that involve viruses.

A New Germ Theory - http://www.theatlantic.com/issues/99feb/germs.htm
The dictates of evolution virtually demand that the causes of some of humanity's chronic and most baffling "noninfectious" illnesses will turn out to be pathogens -- that is the radical view of a prominent evolutionary biologist.

EVA: European Virus Archive - http://www.european-virus-archive.com
Project aiming to create a European network of high calibre laboratories with the expertise to collect, amplify, characterise, standardise, authenticate, distribute and track all viruses.

Viper: Virus Particle Explorer - http://mmtsb.org/viper/
Descriptions of icosahedral virus capsid structures, along with structural and computational analysis.

VirHostNet - http://pbildb1.univ-lyon1.fr/virhostnet
Knowledgebase for virus-host and protein-protein interaction networks.

National Collection of Pathogenic Viruses - http://www.ukncc.co.uk/html/members/ncpv/ncpv.htm
NCPV preserves well-characterised, authenticated human pathogenic viruses in a secure facility. The agents or nucleic acids derived from these viruses are supplied to the worldwide scientific community according to national and international guidelines.

Baculovirus.com - http://www.baculovirus.com
Technical knowledge, information, newsgroup, helpful in using the Baculovirus expression vector system and insect cell expression.

Virology - http://www.elsevier.com/locate/issn/0042-6822
This journal publishes the results of basic research in all branches of virology, including the viruses of vertebrates and invertebrates, plants, bacteria, and yeasts/fungi.

What the Heck is a Virus? - http://people.ku.edu/~jbrown/virus.html
Provides a relatively easy to understand answer to this question.

Virusys Corporation - http://www.virusys.com
Specialists in the design and manufacture of virus related antibodies and antigens. A full range of laboratory services is available that includes infectious dose determination, viral protein purification and custom antibody production.

Blogging for Bacteriophages - http://www.phagehunter.org/
Blog by Tim Sampson who takes a look at recent developments in the world of microbiology.

The Molecules of HIV - http://www.mcld.co.uk/hiv/
The life cycle, immune response, and schematic diagram of the human immuno-deficiency virus.

Plant Virology - http://www.bioinvision.dk/plantvir1.html
Introduction to virus structure, transmission, and transport.

The Phage Phorum - http://www.phages.org
A place to find general information regarding biophages and an open discussion of topics related to biophages.

Veterinary Virology - http://www.pitt.edu/~super1/lecture/lec3351/001.htm
Lecture presentation on the replication of viruses within animals cells.

Viral Replication - http://www.epidemic.org/theFacts/viruses/viralReplication.html
Description of the hepatitis virus replication process.

Medical Virology - http://www.medicalvirology.com
Scientific findings, clinical therapies research, and public health aspects on viruses of medical importance.

GeneJohnson Inc. - http://www.genejohnson.net
HIV genomic data storage and analysis systems. HIVBase allows one to organize HIV sequence data, perform complex statistical analysis, and collaborate data all in one program.

Viralman - http://viralman.110mb.com
Educational site for students provided by H.Niazmand at Azad University of Kazeroun, with downloads and an image gallery.

Hepadnavirus Testing, Inc. - http://www.hvtinc.com

The site describes the services provided by Hepadnavirus Testing, Inc., which uses HBV and DHBV to evaluate disinfectants, inactivation processes, and antiviral drugs for the biotechnology and pharmaceutical industries.

Antiviral Intelistrat - http://www.antiviralintelistrat.com/
Database of antiviral drugs and vaccines. It regroups over 1700 product files containing detailed information on compounds and vaccines related to the prevention or treatment of viral diseases. Subscription needed.

Chicken Flu: Recombinant Genes on the Loose - http://www.synapses.co.uk/science/fluvirus.html
Information regarding viral genes, replication, and strain evolution. Written in 1997 but updated in 2005.

Seminars in Virology - http://www.sciencedirect.com/science?_ob=JournalURL&_cdi=7150&_auth=y&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=7ea28456e36b7099a712a2f126c0c294
Review journal dedicated to keeping scientists informed of developments in the field of virology on a topic by topic basis.

Virus Ultrastructure - http://web.uct.ac.za/depts/mmi/stannard/linda.html
This page is designed primarily as an introductory manual for students of virology,but anyone who is interested is invited to take a brief "peep" at some of the inhabitants of the microbial Lilliput Land.

Journal of General Virology - http://www.sgm.ac.uk/JGVDirect/
Archive of downloadable PDF virus journals.

(Source: Google directory)

Protocols in Virology

Creation and Use of Infectious Virus Vector

http://www.stewartlab.net/Protocols/Making_Virus_1...

(Stewart Lab, Washington University at St. Louis)

Extraction of Geminiviral DNA from a Highly Mucilaginous Plant Protocol http://pubs.nrc-cnrc.gc.ca/ispmb/ispmb18/R00-056.pdf

Protocol for the extraction of geminiviral DNA from a highly mucilaginous plant (Abelmoschus esculentus). Includes: Extraction; Alkali lysis; Cloning.

Generation of recombinant baculoviruses by co-transfection (Bjorkman Group, Howard Hughes Medical Institute at California Institute of Technology)

http://www.its.caltech.edu/%7Ebjorker/Baculovirus-...
For co-transfection prepare at least 10mg of highly purified plasmid DNA. Spodoptera frugiperda cells are sensitive to some contaminants found in crude plasmid preparations, which cannot be removed by phenol/chloroform extraction or ethanol precipitation.

Gradient Purification of AT2 Inactivated HIV-1 Protocol http://ubik.microbiol.washington.edu/protocols/bl3/at2.htm

Protocol for gradient purification of AT2 inactivated HIV-1.

HIV-1 p24 Antigen Capture ELISA Protocol http://ubik.microbiol.washington.edu/protocols/bl3/p24elisa.htm

Protocol for HIV-1 p24 antigen capture ELISA.

Information Resources for Adjuvants and Antibody Production

http://www.nal.usda.gov/awic/pubs/antibody/
Article explaining the function of adjuvants, providing guidelines for their use, particularly Freund's adjuvants, and introducing some alternatives that are commercially available.

Immunohistochemistry: In Situ Hybridization

http://www.immunoportal.com/
Includes details of immunoenzyme techniques, immunofluorescence and in situ hybridizations, with a photograph gallery and discussion board.

Purification of Bacteriophage lambda Arms: Centrifugation through Sucrose Density Gradients Protocol

http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3990

Protocol is used to prepare the arms of any bacteriophage {lambda} vector.

Purification of Bacteriophage lambda Particles by Isopycnic Centrifugation through CsCl Gradients

http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3968

Isopycnic centrifugation through CsCl gradients is used to prepare infectious bacteriophage {lambda} particles of the highest purity that are essentially free of contaminating bacterial nucleic acids.

Purification of Recombinant Adeno-Associated Virus (rAAV) and Parovirus Protocol http://www.axis-shield.com/densityhome/optiprep/M04.pdf

Protocol for the purification of recombinant adeno-associated virus (rAAV) and parvovirus in pre-formed iodixanol gradients.

Retroviral infection of primary human keratinocytes (PHKs)

http://www.methodbook.net/cellcult/infectn.html

(Matt Lewis, Department of Pathology, University of Liverpool)

SDS-PAGE Sample Preparation of Adenovirus Infected Cells Protocol - http://www.bio.com/protocolstools/protocol.jhtml?id=p1752

Protocol for SDS-PAGE sample preparation of adenovirus infected cells.

TEV Purification Protocol http://openwetware.org/wiki/Keating:Experimental_Protocols:TEV_purification

Protocol for TEV purification.

Titration of Retrovirus Protocol

http://www.methodbook.net/virus/titratn.html

Protocol for the titration of retrovirus.

Viral Detection and Concentration in Food http://members.ift.org/NR/rdonlyres/69B323CE-B14C-4DD6-8F56-B25898C9EFB0/0/crfsfsv01n2p082085ms20010312.pdf

Methods and information related to viral detection and concentration in food. Includes: Nucleic acid amplification methods; Recent progress in polymerase chain reaction detection; Detection of human enteric viruses in non-shellfish food; Choice of primers for the detection of human caliciviruses in foods; Biosensors and microarray detection; Alternative confirmation methods.

Virion RNA Dot Blot Protocol

http://www.bio.com/protocolstools/protocol.jhtml?id=p617

Protocol describes the production of a dot blot membrane spotted with RNA isolated from virus particles.

Virus detection protocols for west nile virus in vertebrate and ...

www.ncbi.nlm.nih.gov/pubmed/12904372
Protocols to detect the presence of WNV have been refined since 1999 for ... Polymerase Chain Reaction/methods; RNA, Viral/isolation & purification ...

Virological Laboratory Techniques & Resources

(Source: All the Virology on the WWW)

BioGuide - PCR - general information about the polymerase chain reaction including primer design, hot-start, etc.

BioProtocol, Inc. - This site contains a large collection of high quality protocols for the life science researcher. These protocols include offerings from the virology field of study. The company has a highly reputable editorial board to oversee the quality of its protocol collection as well as an editorial staff committed to procuring and producing the highest quality protocols available on-line.

Detailed protocol for gold labeling proteins for electron microscopy. This protocol describes a modified antibody-protein A-gold technique that eliminates the aggregation problem that frequently occurs with the usual procedure.

Cell Biology Laboratory Manual by Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College, St. Peter, MN.

SPOTTER: a new tool to measure viral plaques. The plaque assay is a classical virological technique, originally developed for bacteriophages by Felix d'Herelle (1917). Dulbecco and Vogt subsequently adapted the assay for mammalian viruses, and it is still widely used for virus isolation and purification, and to determine viral titers. The plaque assay is also frequently used to study in vitro growth properties of mutant viruses. A freeware program,called SPOTTER, operating under Win95 and up, allows convenient and precise analysis of large numbers of plaques.

Protocol On-Line - an exhaustive collection of methods and protocols for molecular and cellular biology from academic and comercial laboratories. There is also a Q&A sections that may be helpful for solving lab technique problems.

Comprehensive Protocol Collection - This resource attempts to offer a comprehensive collection of molecular biology protocols. The site links to protocols either maintained directly by the Ambros Lab at Dartmouth or at other locations around the world.

The HHMI Virtual Lab: This interactive laboratory, developed with Shockwave, demonstrates how an enzyme-linked immunosorbent assay (ELISA) is carried out and some of the experimental problems that may be encountered. A free Shockwave plugin is required.

Molecular Biology Protocols - a General Resource from the NOAA of Molecular Methods, Manuals, and some great Links.

PCR in situ: New Technologies with Single Cell Resolution for the Detection and Investigation of Viral Latency and Persistance" from the University of Minnesota

PCR Jump Station - This site attempts to provide scientists with a comprehensive directory for the polymerase chain reaction (PCR) and related techniques.

Labexplorer.com is a new web site written by clinical laboratorians for clinical laboratorians. Designed to be a comprehensive resource for those interested in clinical laboratory practices, the site provides a mechanism of communication between laboratorians, access to experts in a variety of clinical fields, job and career information, and comprehensive links to laboratory sites, diagnostic companies, organizations & interesting topics.

Molecular Biology Protocols on the Web

a great collection of protocol links from the publishers of Molecular Biology Today.