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N-terminal and Internal sequences of proteins in gel

Amino acid sequence information can be obtained quickly if you provide us with protein in a small piece
of SDS-PAGE gel.

N-Terminal sequence
The initial yield of samples prepared by SDS-PAGE and electro-blotting onto PVDF may be as low
as 5-30% (see also our separate information). Furthermore, such a strategy of sample preparation
is not very suitable if several spots from 2D-gels have to be combined to generate sufficient sample.

Therefore, we offer a service to isolate the protein(s) of interest from a piece of gel, followed by
sample clean-up and sequence analysis. In our hands, this method has proven to be superior in
'initial yields' compared to standard SDS-PAGE and blotting procedures. Moreover, extracts from
several spots of 2D-gels can now easily be combined to increase the quantity.

For this analysis, standard gels (with a maximum thickness of 0.5-0.75 mm) and Coomassie Brilliant
Blue staining procedures can be used (see for instance protocol mentioned below). At the moment,
a minimum quantity of about 10  pmol of protein is required, but higher quantities, e.g. 50 pmol or
more, are preferred. Please consult us in advance if you intend to send less than 10 pmol.

Internal sequences
Internal sequences are amino acid sequences of a protein other than its N- or C-terminal sequence.
Since many proteins are not susceptible to Edman degradation due to N-terminal blocking, and no
routine direct methods exist for reliable C-terminal sequencing, sequence information of the protein
has to be derived from its internal regions. This is usually done by N-terminal sequence analysis of
a peptide obtained after digestion and purifica­tion from a protein digest. Such a strategy is also
followed in case it is not known whether the N-terminus is blocked and one does not want to take
the risk that no sequence information will be obtained after N-terminal sequence analysis of precious protein material. Another reason would be that protein identification by peptide mass fingerprinting
failed or is assumed to fail because hardly any DNA or protein sequences are available in the public
data bases for that particular protein / organism. 

Internal sequencing of a protein present in a piece of SDS-PAGE gel (or 2D-gel) is performed by an
in situ tryptic digestion of the protein, followed by extraction, RP-HPLC purification, selection and
sequence analysis of (a) suitable fragment(s). In our hands, this method has proven to be successful
for many hundreds of proteins analyzed so far. Moreover, we offer specific selection of Trp-containing peptides for sequencing, if the design of a proper DNA probe is of importance (unique codon use of Trp).
After the extraction of the peptides, a check is performed. If the results indicate a successful digestion
and the presence of enough peptide material, the subsequent steps will be performed. If no sufficient signals of internal pept­ides are found, we will perform an additional digestion, free of charge, after we have received another piece of gel with more material. Also, we may consult you about other aspects,
if choices have to be made.

It is possible to obtain sequence information from small quantities of protein and to take full advantage
of the high resolution power of SDS-PAGE. We only ask you to provide us with protein in a piece of gel (see below for sample preparation). Although it is possible to obtain sequence information from as little
as 5-10 pmol of protein (= 0.5 - 1 µg of a 50 kDa protein), higher quantities e.g. 50 pmol or more, are preferred. Please consult us in advance if you intend to send less than 50 pmol. Usually, sequence information will be available within 2-3 weeks after you have provided us with the sample.

When preparing your sample, please mind the following

  • a high protein/gel ratio is important. Therefore, please use a gel with a thickness of 0.5-0.75 mm and prepare at least 50 pmol of protein material in preferably one, as small as possible, piece of
    gel. Please consult us in advance, if you are planning to send less than 50 pmol.

  • Standard CBB (Coomassie Brilliant Blue) staining (0.1% CBB in 45% methanol, 10% acetic acid;
    30 min. at room temperature) and de-staining (45% methanol, 10% acetic acid; 30 min. at RT) procedures can be used.

  • When cutting the gel slice, please cut at least one mm around the visible band, if possible.

  • The piece of gel should be sent to us as quickly as possible. If stored, please store at ±4oC.

Samples can be sent to us by mail or courier, for instance in a sealed eppendorf tube. Some additional protection against damage is advised. If a long postal time is expected, cooling at  ±4oC is
recommended. Please, fill in our sample submittal/order form for sequence analysis, or at least inform
us about the Mw of the protein and your estimation of the quantity present in the piece of gel. Also,
let us know if you wish a positive identification of cysteine residues (optional).

If we are not able to obtain sequence information, we will charge a reduced price.