Welcome

The Common Protein Sequencing Facility
Contact
Sample requirements
Results
Order form
N-terminal sequencing
Amino acid analysis
Price list

The Common Protein Sequencing Facility
 
 

The Common Sequencing Facility in Lyon is located in the Institut de Biologie et de Chimie des Protéines (Lyon-Gerland). It's part of CCMP facilities from IFR 128.

Our purpose is to provide protein analysis solutions for research teams.

We offer the following services :

N-terminal sequencing     SEQ
Amino Acid Analysis     AAA
 
 

Contact

Dominique Mazzocut

IBCP-Centre Commun de Séquençage
7, passage du Vercors  69367 LYON Cedex 07

Tél. +33(0)4 72 72 26 63            Fax. +33(0)4 72 72 26 02
e-mail : seq-aaa@ibcp.fr

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Sample requirements

Samples should be submitted according to the following :

Pure samples, either dry, in solution (water, TFA, acetonitrile) or blotted onto PVDF membranes (we recommend high binding membranes : ProBlot, Immobilon Psq or equivalent with Coomassie Blue R250 or Ponceau S staining).

For samples in solution, the volume should be < 50 ml with no salts or detergents

Samples must be shipped in an Eppendorf-like tube and clearly labelled
 
 

About your results

Analysis reports will be sent by mail or e-mail. Graphs, data sheets and validation data may be attached upon request.
 
 

Order form

Order forms should be sent directly to :
IBCP-Centre Commun de Séquençage
UMR 5086 CNRS-UCBL
7, passage du Vercors
F-69367 LYON Cedex 07
 
 

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N-terminal sequencing

Protein or peptide N-terminal sequencing is carried out by automated sequential degradation aka Edman chemistry.

Chemistry

Edman chemistry consists of 3 major steps :
 

1.  PITC (phenylisothiocyanate) coupling at the N-terminus (pH 9-10), giving a PTC (phenylthiocarbamate) group
2. Cleavage with TFA (trifluoroacetic acid) and extraction of an ATZ (anilinothiazolinone) amino acid derivative
3.  ATZ conversion under acidic conditions to a more stable PTH (phenylthiohydantoin) amino acid derivative

During these steps, reagent excess and reaction by-products are washed out.

PTH derivatives are then identified by on-line microbore reversed-phase HPLC.
 

Equipment

Our facility operates two automated sequencers from Applied Biosystems : Procise 492A and 473A. These systems permit analysis in the range  1-100 pmoles (Procise) or 50/500 pmoles (473A). We can treat samples in solution as well as PVDF blotted one. For specific runs, fraction collection is available.

Integrated software permit chromatogram analysis and help in sequence calculation.

Yields and reading length

Sequencing performance for a specific sample depends on initial yield, repetitive yield, lag (or carry-over) and background noise.
Initial yield (typically 30-60%) is the amount of the first residue of a run expressed as a percentage of the total protein amount.
Repetitive yield (generally 90-98%) shows the amino acid ratio from cycle to cycle. It depends on both the instrument performance and the sample itself.
Lag or carry-over is the amount of amino acid from cycle n still present at cycle n+1.
Background noise is due to incomplete cleavage or internal cleavage in the sample. This noise increases cycle after cycle.

As a result of these factors, sequence reading will not be possible once the background is higher than the actual amino acid signal. Depending on samples and on the amount of protein, the length of sequence reading can vary from a couple of residues to several tens of residues.

Helpful web page :
http ://www.hsc.virginia.edu/research/biomolec/seqguid4.htm
 
 

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Amino acid analysis

After complete hydrolysis, amino acid analysis consists of quantitation of all amino acids from a sample.
This gives us both qualitative (composition) and quantitative (amount) informations.
 
 

Equipment

Our facility uses post-column derivatization (ninhydrin derivatization after IEX HPLC) on a Beckman 6300 instrument handling down to 10mg of protein.
Complete hydrolysis is carried out under vacuum with a vapor HCl-TFA mixture on a Waters PicoTag Workstation.
After analysis and integration, results are presented on a dedicated MS-Excel data sheet.

Helpful web page :
http://www.abrf.org/ABRF/ResearchCommittees/aaaarticles/aaafiles /aaa.html
 

Price list (VAT not included)

 

SEQ:          start-up : 210  €           per residue : 45 €

AAA:           per sample :  95 €

 For CNRS laboratories please contact us.
 

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