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Protein Sequencing - Technical Details

For protein sequencing, we used N-terminal Edman degradation chemistry. Our high-throughput capability ensures the fastest possible turnaround time for providing you with the amino acid sequences of your samples.

The Protein/Peptide Sequencing Capability

Guidance to choose the required number of degradation steps

Sequencing Method

The process uses the very well established technique of Edman degradation.

Here, one amino acid at a time is removed from the N-terminal of the protein by alternately reacting the protein with the Edman reagent, (phenyl isothiocyanate, PITC ) and cleaving the resulting compound off the protein with acid. Identification of the amino acids is achieved by their elution times on HPLC compared to a standard mixture. 

The protein is immobilised inside the instrument by either blotting it first onto a PVDF membrane or by its adsorption onto a biobrene treated glass fibre filter. The cleaved amino acids are converted to their PTH derivatives, ( phenylthiohydantoin) before being automatically injected onto the onboard HPLC instrument. Data from the HPLC is collected on a computer for visual calling of the sequence.

Sample Preparation

Samples should contain a minimum of 2 picomoles of the protein/peptide to be sequenced. Protein can be submitted dry, in solution, as an SDS or native polyacrylamide gel piece, or blotted to PVDF membrane.

On PVDF Membrane

In general, best results are obtained using 2D gels blotted to PVDF we can perform the blot for you if you wish to supply sample on 2-D gel. The protein can be stained with Coomassie Blue R-250, Amido black, Ponceau S or Sypro Ruby. Do not use silver stain. NB Coomassie Blue R-250 detects protein by mass (from 30-40 ng per band). A moderately intense spot of 10kDa containing 100ng protein will have a concentration of 10 picomole. At 100kDa a post of the same intensity will only contain 1 picomole. Coomassie Blue G-250 is more sensitive than R-250 and protein is comparatively more visible, therefore insufficient protein could be available for sequencing when stained with G-250. Nitrocellulose and nylon membranes cannot be used as they are unable to withstand the sequencing conditions and cause severe damage to the sequencing apparatus.

A whole PVDF blot may be submitted dry, between two sheets of blotting paper, and sealed in a plastic bag. Indicate the appropriate bands to be sequenced on a labelled photocopy of the membrane (Please do not mark the blot with ink or pencil. Bands may be outlined by making holes with a sterile needle).

On Gels

Protein can be passively eluted from polyacrylamide in an overnight procedure for an additional fee. Gels should be stained with Coomassie Blue R-250 or G-250. Do not use silver stain. Note: Passive elution is generally less efficient than electroblotting and does not work with high mw proteins. It is recommended for well stained protein bands that are less than 60kDa.

Considerations

Sequencing Reads

No sequence will be seen if the protein is N-terminally blocked either naturally (e.g. by formyl, acetyl or pyroglutamyl groups) or accidentally during isolation and storage (see above Note regarding in solution samples).

Cys will give a blank result unless reduced and alkylated Acrylamide is the recommended alkylating agent. Specify on request form how Cys was modified.

Post-translationally modified amino acid residues (e.g. glycosylated or phosphorylated) will give a blank cycle upon sequencing. Identification of these residues may be possible by running modified methods on the sequencer.

Sample Purity


Sequencing is often limited by an inability to obtain sufficient amounts of adequately purified protein. Samples should contain one protein component only and reagents which interfere with the sequencing process should be avoided. The presence of contaminants increases the likelihood that ambiguous data will be obtained and the chances of miscalls are greater. Clean samples tend to yield better results and can be sequenced further. Contaminating peptides or proteins contribute to a higher noise level of non-sequence related amino acids.

Protein Quantity

We can read sequences with only 2-5 picomoles of protein, although this is at the extreme limit of detection. 20 - 50 picomoles is a more realistic amount. A rough and ready guide is if you can photocopy the bands then they can be sequenced. Scans tend to enhance the blot.

The image on the left shows a typical PVDF blot.

Bands 1-5 would need several lanes in order to get enough signal and would be at the limit of detection.

Bands 6 and 7 would give a nice signal with just one band on the sequencer.

There is not enough material in band 8.

Band 9 would give a strong signal and is about 50pmole.

For analysing peptide libraries, a peptide on a single bead gives a very strong signal.

 

 

N-Terminal Blockage


Proteins may be blocked naturally, as in eukaryotic cells (at a rate of 70% or greater), or they may be blocked during the process of purification. In the latter case protocols may be altered in order to avoid blockage. If this is not possible, one may attempt to deblock the protein or try internal sequencing.

Blocked samples result in chromatograms that show no specific amino acids but will display a gradually rise in of all or most of the amino acid peaks with each successive cycle of the Edman chemistry.

GLP/GMP Submissions

All data is archived for at least 10 years and the facility is run to GLP/GMP standards.