Bioprotocols: 2010-01-10

Methods of Telomerase Inhibition
Additionally, methods are outlined in this chapter for determining the effectiveness oftelomerase inhibition through TRAP assays or assessment of telomere ...
www.ncbi.nlm.nih.gov/pmc/articles/PMC2423206/

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

Protein Purification Related Articles

Related Articles

Chromatographic Protein Purification Methods

Protein Shakes for Kids

Protein Function

Bodybuilding Nutrition Advice - Lee Labrada Talks About Mr. Olympia Bodybui...

Easy Low Carb Menu - High Fiber High Protein Low Carbohyd

Related Articles

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
Komala Ponniah, Trevor S. Loo, Patrick J.B. Edwards, Steven M. Pascal, Geoffrey B. Jameson, ...

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™
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 6 December 2009
Cornelia Ludwig, Martin A. Wear, Malcolm D. Walkinshaw

5. Expression, purification and primary crystallographic study of human androgen receptor in complex with DNA and coactivator motifs
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 6 December 2009
X. Edward Zhou, Kelly Suino-Powell, Phumzile L. Ludidi, Donald P. McDonnell, H. Eric Xu

6. Purification and characterization of recombinant CH3 domain fragment of the CREB-binding protein
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 6 December 2009
Catherine Ibarra Drendall, Quang H. Pham, Eric C. Dietze

7. Establishment of a simple and rapid method to screen for strong promoters in Bacillus subtilis
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 4 December 2009
Trang Thi Phuong Phan, Hoang Duc Nguyen, Wolfgang Schumann

8. An Improved Procedure for the Purification of the Escherichia coli RNA Polymerase Omega Subunit
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 4 December 2009
Catherine E. Vrentas, Tamas Gaal, Richard R. Burgess, Richard L. Gourse

9. Establishment of a simple and rapid method to screen for strong promoters in Bacillus subtilis
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 4 December 2009
Trang Thi Phuong Phan, Hoang Duc Nguyen, Wolfgang Schumann

10. An Improved Procedure for the Purification of the Escherichia coli RNA Polymerase Omega Subunit
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 4 December 2009
Catherine E. Vrentas, Tamas Gaal, Richard R. Burgess, Richard L. Gourse

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