2 rue des Gravilliers - 75003 Paris - France
Tel. : +33 142 71 70 21 - Fax : +33 142 71 70 23
email : info@genosphere-biotech.com

Protein Sequencing

Welcome to our Bioanalysis Division. Whether you are trying to characterize an unknown protein sample (15 to 20 residues sequencing) or simply require sequence confirmation of an expressed protein (5 residues sequencing), this is the service you need. Should you have any questions or requests, please don't hesitate and contact us. N-terminal protein sequencing by Edman degradation approach Up to 25 residues BLAST search results & analysis report including original chromatograms

Service Offer

N-terminal protein sequence analysis
This procedure involves the sequential (i) chemical modification of the N-terminal residue, through its amino group, (ii) removal of NH2-derivatized amino-acids from the N-terminal to the C-terminal of a peptide chain, and (iii) reverse phase HPLC analysis with UV detection at 270 nm. Comparison of the successive elution profile to that of a standard set of adducted amino-acids allows for sequence calls (Fig.1).

Fig. 1. HPLC elution profile of a standard mixture of 19 adducted amino-acids providing standard retention times. Several by-products, indicated by an X, are formed during the Edman degradation process.

Other Services

• Internal sequencing by chemical cleavage: please submit your project details for evaluation.

Sample Requirements

-Sample amount: 15 to 20 pmol

- Sample form
Blotted onto a PVDF membrane (Preferred)
The protein should be electroblotted from 1D or 2D gels and stained with Comassie Blue (R250). Following staining/destaining, blotted membranes should be rinsed thoroughly with deionized water. The sample should be as concentrated as possible (15 to 20 pmol / lane) and several gel lanes may be loaded and submitted. The whole membrane may be submitted with a copy showing the band(s) of interest or the bands may be cut out and submitted, please indicate band molecular weight.

In solution or lyophilised (we will run pre-purification at nominal fee)

30 to 150 µl of solution (H2O, Acetonitrile, Propanol..). Pure lyophilised protein is also acceptable.

Protein should be free of detergent, salts and buffers. Please note that any amine-containing chemical present (e.g. Tris) will impair the adduction step.

-Sample purity

PVDF blots: well separated bands.
Liquid: Single purified proteins. Immunoprecipitation & Affinity-column purified usually give good samples for sequencing. In any case, a mandatory additional clean up is applied to all incoming liquid samples.

Inherent Technical Limitations

• Peptides & proteins with blocked N-terminal amino groups (e.g. acetylated, formylated) will not sequence by Edman degradation.

• High molecular weight proteins (over 75 kDa) may prove difficult to be analyzed. In our experience, the foremost reason is sample quantity and / or purity. However, reasonably pure protein bands and comfortable amounts will allow for unambiguous sequence calls. Please contact us before sending your sample.

• Unmodified Cys and glycosylated residues will give a blank cycle (i.e. residue is cleaved off the peptide chain but is not detected by HPLC at 270 nm).

• Sample quality is critical. Any impurity that interfers with Edman's chemistry (mainly amine-containing chemicals) will lead to poor results.

Ordering Guideline (see also General Ordering Information)

You may find our pricing table at: Pricing

A set up fee (minimum charge) that includes the first 5 residues applies.

Please send your PVDF membrane(s) or tube(s), clearly labelled and wrapped with parafilm in a padded envelope. Include your Institutional Purchase Order (mandatory) and a letter describing your sample(s) and indicating the number of residues you wish to sequence. Should you require a database search, please indicate protein details (species, suspected family, etc.).

PVDF Samples: Copy marking band(s) of interest, Approximate size, Amount loaded.

Solution Samples: Sample name, Approximate size, Total amount (µg), Concentration (µg/µl), Volume (µl) and solution content (H2O, buffers...).

Please send using express delivery courier or postal services at:
GENOSPHERE BIOTECHNOLOGIES
Protein Analysis Service
2 rue des Gravilliers
75003 PARIS
FRANCE

Receipt of your samples is confirmed to you by e-mail and an approximate date of completion is released to you.