Fee-for-Service: N-Terminal Protein Sequencing

N-terminal Edman sequencing of proteins & peptides

We provide
N-terminal sequencing of proteins and peptides provided as lyophilized samples, in solution or blotted on PVDF membrane as fee-for-service. Proteins and peptides can be sequenced five to ca. 40 steps depending on sample amount, amino acid sequence and customer’s requirements.

You receive
a result report of the N-terminal sequence analysis with the detected amino acid sequence and all HPLC chromatograms as a PDF file by email or via one of our SSL and password protected web servers.


N-terminal proteins sequencing using a Procise Edman Sequencer

Application area for protein sequencing

Determination the N-terminal amino acids of a protein
Determination the N-terminal amino acids of a peptide
Check of the correct translation of a recombinant protein
Check of the sequence and purity of a recombinant protein
Check of the sequence and purity of a synthetic peptide
Determination of complete protein sequence in combination with additional methods
Quality control



How to order
Please contact us at first. We can give you some helpful information and send you our protein Edman sequencing sample form. You can also download our N-terminal Edman amino acid sequencing sample submission form here. Then ship the sample together with the filled Edman sequencing sample form to us.
Below you find information about sample requirements and shipping.


 
 
 

Sample requirements

Probenmenge  mind. 20 pmol, geringere Mengen werden nur nach Rücksprache akzeptiert!  
Sample type  1. lyophilized protein/peptide samples
2. liquid protein/peptide sample in 10-100µl volatile solvent like Milli-Q water. isopropanol, acetonitrile. The sample have to be shipped in frozen state!
3. Protein samples blotted on PVDF membrane (PVDF membrane size: max. 3x6 mm, smaller and more concentrated sample are preferred. The protein band can be stained by Ponceau S, Amino black, Sulforhodamine or Coomassie Brilliant Blue.  
Purity  The sample should be as pure as possible (at least 75-80% purity) and contain only the protein or peptide. Free amino acid, primary amines, SDS, salt, buffer or other contaminants should be as low as possible since the Edman chemistry can be negatively affected. 
Cysteine modification   Cysteine without special modification can not be detected by N-terminal sequencing. Therefore the sample has to be modified for detection of cysteine. Below you can find a modification protocol. 
N-terminal blockage  Proteins and peptides which are N-terminally blocked do not have a free N-terminal amino group. Therefore these proteins can not be sequenced. More than 50% of all eukaryont proteins are blocked. On request we can perform deblocking procedures but we need a significant higher amount of protein and the deblocking does not work always, because mostly the type of blockage is unknown. 
Glycosilation and other modifications   Edman sequencing steps without the detection of an PTH amino acid, reduced peak intensity or altered retention times can be caused by glycosilation, phosphorylation or other modifications. These amino acids can not be sequence often. By mass spectrometry the modification can be analyzed. 



Details for sample shipping to Proteome Factory

Please, fill the Edman protein sequencing form with information about the samples (e.g. sample name, sample type, MW, protein amount, staining method) and the requested number of N-terminal sequencing steps. Sign the form!
Send us an email or call us before shipping the samples
The samples should be in an Eppendorf reaction tube sealed by Parafilm. Protein samples on PVDF membrane can either be shipped as small and dry membrane slides in reaction tubes or as a dried PVDF membrane in plastic foil with enclosed description of the protein bands which should be analyzed. We cut the marked protein band for analysis.
Liquid samples should be sent in frozen state.
Lyophilized sample and samples blotted on PVDF membrane can typically shipped at room temperature.
Please, send your samples in a padded envelope or in a box together with the Edman protein sequencing form.



Our Recommendation for Protein Blotting



Semidry PVDF Blotting Protocol

We recommend the following protocol for semidry blotting on PVDF membrane:

- PVDF membrane: Immobilon P membrane or comparable

- Blot buffer: 50 mM sodium borate, pH 9.0 / 20% methanol (HPLC quality)
(0.1% SDS can be added to blotting buffer if protein above 40kDa should be sequenced)

- Blotting condition: 1mA/cm1 PVDF membrane for 2-3 hours at 4°C
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Protein staining of PVDF membranes

Ponceau Red Staining

Staining solution (0,5% Ponceau S, 1% acetic acid in Milli-Q water):

0,25 g Ponceau S
0,5 mL acetic acid
ad 50 mL Milli-Q water

Procedure:
1. Wash the PVDF blot membrane 2x 3 minutes with plenty Milli-Q water.
2. Stain the PVDF membrane with Ponceau S staining solution for 1-3 minutes.
3. Destain the PVDF blot membrane under visual control with Milli-Q water until protein bands are well visible.
4. Dry the PVDF membrane.

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Sulforhodamine Staining

Staining solution (0,005% sulforhodamine, 0,2% acetic acid, 30% methanol in Milli-Q water):

150 mL methanol
1 mL acetic acid
25 mg sulforhodamine
ad 500 mL Milli-Q water

Procedure:
1. Wash the PVDF blot membrane 2x 10 minutes with plenty Milli-Q water.
2. Dry the PVDF membrane at room temperature!
3. Stain the PVDF membrane in Sulforhodamine staining solution for 1-2 minutes.
4. Wash the PVDF membrane with Milli-Q water shortly.
5. Dry the PVDF membrane.

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CBB R250 Staining

Staining solution (0,1% CBB R250, 10% acetic acid, 40% methanol in Milli-Q water):

0,1 g CBB R250
40 mL methanol
10 mL acetic acid
ad 100 mL in Milli-Q water



Destaining solution (10% acetic acid, 40% methanol in Milli-Q water):

40 mL methanol
10 mL acetic acid
ad 100 mL Milli-Q water

Procdure:
1. Stain the PVDF blot membrane for 5 minutes in CBB R250 staining solution
2. Destain the PVDF blot membrane for 3 x 5 minutes with destaining solution under visual control until protein bands are well visible.
3. Dry the PVDF membrane.

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