Biocentrum - Sequencing of proteins and peptides

Sequencing of proteins and peptides

Our studies

Determinations of protein and peptide sequences are carried out using Edman chemistry on Applied Biosystems model 491 automatic sequencer. To better realize the specific expectations the analyses are performed in a close electronic contact with client.

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Important information considering protein sequencing

Proper preparation of samples is of principal importance for successful sequencing. In case of any doubts or questions please contact us before sending a material.

The automatic sequencer analyses two kinds of samples: the liquid ones and the PVDF membrane-bound ones. The liquid samples are absorbed on a special glass-fiber filter before sequencing while the membrane-bound samples are placed directly in the sequenator sample cell.

The liquid samples should contain minimum 50 picomols of a homogenous protein or peptide in a volume of maximum 50 microliters. The above quantity of 50 picomols is enough for determination of sequence of over a dozen of amino acids, depending on sample purity and its molecular mass. The very long sequences (30-50 residues) require about 1 nanomol of sample. The sample should be dissolved in ultra-pure water. The other solvent should have maximal ionic strength of 0.05 Mol/liter. The solvents have to be free from primary amine salts, tris, glycine, ampholines etc. No glycerol and detergents should be added. The simple inorganic salts, as sodium phosphate or sodium chloride are acceptable. The best way to proper purify and desalt a protein or peptide sample before sequencing is performing the HPLC purification step on a C-8 or C-18 column, using volatile buffer systems (e.g. water/acetonitrile, TFA). The collected into methanol-washed tube homogenous fraction should be lyophilized, sealed and shipped for analysis. In case of lyophilization of salt-containing samples please observe that the obtained powder will be dissolved in about 50 microliters of water and the final ionic strength of this solution should be less than 0.05 Mol/liter.

In case of membrane-bound samples only PVDF membranes are accepted (no nitrocellulose or nylon). If possible, please use special-purpose sequencing-grade PVDF membranes (as Immobilon-PSQ from Millipore or Sequi-Blot from BioRad). During electrotransfer of proteins from SDS-PAGE gels please use CAPS buffer, no tris-glicine buffer is allowed. Because silver-staining is not compatible with sequencing please stain the membranes using Coomassie Brillant Blue, Amido Black or Ponceau S. After staining the membranes should be washed in HPLC-grade water and allowed to completely dry. Please mark the interesting bands by circles and ship the whole membrane, we will cut out the bands by oneself. If this is impossible please cut out the interesting bands using clean, sharp blade. Please observe that membrane pieces should be as small as possible. The sequencer cell accepts membrane pieces of maximal dimensions 0.5x1.0 cm but the best results can be obtained during sequencing of small sharp stripes of 0.2x0.5 cm dimensions. The amount of proteins or peptides absorbed on membranes should be equivalent to awaited sequence length: the 50 picomol amount will allow to obtain about 10 amino acids residues, while 200 picomols may allow to identify about 30 residues. The sequencing of PVDF membrane-bound samples gives best results in case of small proteins (in range from 2 to 50 kDa). The proteins above 50 kDa should be sequenced rather from liquid-phase. In calculation of a total amount of protein absorbed on membranes please keep in mind the fact that electrophoresis, electrotransfer and staining processes lose about 50% of primary material loaded on the gel. The membrane ready for shipping should be carefully packed in original membrane spacers or in filter paper and expedited in cardboard envelope to avoid mechanical damages.

ATTENTION: If not otherwise stated by the client, the samples will be disposed after analysis.

In publications please use the following description of the procedure: "N-terminal protein sequence analysis was performed at BioCentrum Ltd. (Kraków, Poland). The sequentially detached phenylthiohydantoin derivatives of amino acids were identified using Procise 491 (Applied Biosystems, Foster City, CA, USA) automatic sequence analysis system working according to a standard protocol of manufacturer."