Biotech Articles
Publish Your Research Online
Get Recognition - International Audience

Request for an Author Account   |   Login   |   Submit Article
 
 
HOME FAQ TOP AUTHORS FORUMS PUBLISH ARTICLE
 
 

Edman Degradation - For Protein Sequencing

BY: Muniba Safdar | Category: Genetics | Submitted: 2010-08-10 06:15:56
       No Photo
Article Summary: "What is Edman Degradation method? It is a chemical method used for the sequencing of amino acids in a peptide. This method is developed by Pehr Edman in 1960. In this methods, N-terminal or amino terminal is labeled or cleaved from the peptide and number of residues are identified..."


Share with Facebook Share with Linkedin Share with Twitter Share with Pinterest Email this article
     


Edman degradation is a chemical method used for the sequencing of amino acids in a peptide. This method is developed by Pehr Edman in 1960. In this methods, N-terminal or amino terminal is labeled or cleaved from the peptide and number of residues are identified.

Process
• Suppose we have a sequence MYKMRYY.
• We will label amino terminal with isothiocynate (component of certain plants and vegetables).
• Then we will do mild hydrolysis, on hydrolysis only labeled amino acids will be separated.
• Through HPLC, High Pressure Liquid Chromatography, we will identify this labeled amino acid.
• Then again label the N-terminus of remaining polypeptide chain.
• All the above steps will be performed for all amino acids.

Disadvantage of Edman Degradation

• It is very exhaustive method.
• Start with N-terminus, if protein not linear and N-terminus inward or if N-terminus is modified then reagent will not bind, we will not proceed.
• In 24 hours only 10 residues identified.
• More protein sample is required.
• Upto 50-60 residues this process is efficient and accurate, above this, its efficiency reduced.

Advantage of Edman Degradation


• 100 % surety of finding protein sequence.

To solve or increase efficiency

• To solve or increase efficiency we will fragmentize whole polypeptide.
• Suppose we have 400 peptides, fragmentize this 400 peptides, using different enzyme like trypsin. It cuts at basic amino acid (Arginine/ lysine) if next amino acid is not proline.
• Arginine/lysine amino acid exists commonly.
• In 500 amino acids protein, there is chance of 20-25 sites to fragmentize.
• So all fragments will be of different and unknown size.

Problem: We do not know about the arrangement or sequence of these fragments.

Two ways to solve problem

• Uses different reagents to cut the peptides/multiple proteases.
• Fragment assembly or assemble fragment.

Assemble fragments

One sequence is digested with trypsin. After digestion these fragments generates.

1. Lue-Asp-Glu-Tyr-Gly-Val-Ile-Lys.
2. Ala-Val-Ile-Leu-Ser-Glu-Ile-Leu.
3. His-Thr-Val-Glu-Val-Arg.


The same sequence will be digested now with Glu-C. The following fragments will be generated.

1. Leu-Asp-Glu.
2. Tyr-Gly-Val-Ile-Lys-Ala-Val-Ile-Leu-Ser-Glu.
3. Ile-Lys-His-Thr-Val-Glu.
4. Val-Arg.


By overlapping these fragments we will find out which fragment will be next for example compare fragment of trypsin digestion and fragment of Glu-C digestion. We will know first three amino acids.

Overlapping sequences (fragments) can be sequenced following digestion of the protein with a reagent with different specificity to trypsin. It is important to choose a reagent with a very long and specific recognition site.

The solved amino acid sequences of peptides can be used to design degenerate PCR primers which can be used to isolate a corresponding genomic or cDNA sequence. This can then be translated to predict the full length protein sequence.

About Author / Additional Info:


Search this site & forums
Share this article with friends:



Share with Facebook Share with Linkedin Share with Twitter Share with Pinterest Email this article

More Social Bookmarks (Digg etc..)


Comments on this article: (0 comments so far)

Comment By Comment

Leave a Comment   |   Article Views: 32615



Additional Articles:

•   Artificially Induced Mutations

•   Bacillus - Industrial Work-Horse of the Microbial World

•   A List of Bioinformatics Journals

•   Molecular Farming: Agriculture with value added harvest




Latest Articles in "Genetics" category:
•   The Science and History of Genetics. How It Predicts the Genetic Code

•   Telomeres: Is It Responsible For Ageing and Cancer?

•   Human Genetic Engineering,its Methods and Ethics

•   Gene Mutation And Cancer

•   DNA Technology Used in Forensics

•   DNA Fingerprinting: Uses and Methods Involved

•   Treatment of Genetic Diseases by Gene Therapy

•   Human Intelligence and Genetics

•   Ethical Issues Related to Human and Animal Cloning

•   Mitochondrial DNA and Forensic

•   DNA Footprinting and Gene Sequencing

•   Biotechnology and Types of Cloning

•   Designer Babies:Method and Ethical Issues

•   Prenatal Diagnosis: Non-invasive and Invasive Techniques

•   What are the Benefits of Genetic Engineering?

•   The Advantages and Disadvantages of Genetic Engineering in Humans

•   Types of Genetic Disorders

•   Bovine Somatotropin: A Growth Hormone

•   Advantages and Disadvantages of Genetically Modified Food



Important Disclaimer: All articles on this website are for general information only and is not a professional or experts advice. We do not own any responsibility for correctness or authenticity of the information presented in this article, or any loss or injury resulting from it. We do not endorse these articles, we are neither affiliated with the authors of these articles nor responsible for their content. Please see our disclaimer section for complete terms.
Page copy protected against web site content infringement by Copyscape
Copyright © 2010 biotecharticles.com - Do not copy articles from this website.

ARTICLE CATEGORIES :
Agriculture Bioinformatics Applications Biotech Products Biotech Research
Biology Careers College/Edu DNA Environmental Biotech
Genetics Healthcare Industry News Issues Nanotechnology
Others Stem Cells Press Release Toxicology  


  |   Disclaimer/Privacy/TOS   |   Submission Guidelines   |   Contact Us