Q. How should I present the sample?

We can work with

proteins in solution
on PVDF blots
as gel slices

A protein on a Blot is the easiest method. The instrument can work with 2 or 3 bands worth of blot, (or about 5mm x 5mm in area).

Proteins in a gel can be transferred onto a blot for a small extra charge. One advantage of this method is that any amount of gel slices can be used, useful when the protein is present in a large number of lanes.
Please don't send whole gels, as they tend to break up in transit.
Just send the gels slices as they are, don't suspend in water as the protein will diffuse out.

Please do NOT send the sample on nitrocellulose blots. Nitrocellulose degrades in the sequencer and gums up the tubing.

QHow many amino acids will I need to read?

For many applications long sequence reads are not required.

If the peptide or protein just needs to be confirmed as part of a known sequence, then about 5 or 6 amino acids are all that are needed.
For a database search, about 10 amino acids are wanted.
To get enough data to make a DNA probe will require from 10 to 15 amino acids to ensure the choice of a suitable primer with minimal degeneracy.

QHow long will I have to wait to get my data back?

Typically the turnaround time is 2-5 days, depending on the workload at the time.

QHow much protein will I need?

QWhat is the best blot material?

The blot must be PVDF, (nitrocellulose will kill the machine).
Material such as Pro-Blot from PE Biosystems or Immobilon-Psq from Millipore work very well.

QWhat is the best way to stain a blot?

We recommend the use of Amido Black, and Ponceau yellow ( or red ) as stains, these seem to give the best results by far.
Commassie Blue will work but some grades are thought to interfere.
High sensitivity stains will work but they will indicate bands that are far below the level of detection.

Don't use:- Gold, Silver, Antibodies. Gold and silver appear to block the protein, and we would end up sequencing the antibodies.

QWhat is the best way to obtain protein fragments for sequencing?

When generating internal sequences from a protein, a cyanogen bromide digest is advised over enzymatic methods such as trypsin. Trypsin usually gives a large number of peptides which can be very difficult to resolve on HPLC and are usually too small to separate on a gel. A CNBr digest tends to give a few large fragments which can be readily separated on a gel.

QWhy can't I get a sequence from my band?

About half of the Eukaryotic proteins have a group on the N-terminus which prevents any Edman sequencing.
There is little that can be done to remove these compounds and the way forward is to attempt an internal sequence
If the band is faint, then it is difficult to say whether the fault is due to insufficient material or that the N-terminus is blocked.

QAre there any things to take special care about?

Try and avoid glycine or Tris based buffers in the stages of purification. Glycine cannot be completely removed afterwards and will appear in the first one or two cycles of the sequence, masking any sequence glycine.
When working at high sensitivities, blots give far better backgrounds than free proteins obtained from HPLC. Blot material should be polyvinylidene difluoride, PVDF, (trade names such as ‘Immobilon PSQ’ or ‘Pro-Blott’), (nitro-cellulose is NOT suitable). However blots do not give as long a sequence read as proteins in solution. The instrument can take a maximum of 25sq.mm of blot. In general, the lower the area of membrane the lower the background.
Use volatile buffers for purifying proteins on HPLC. The sample volumes will probably have be reduced before sequencing, (should be less than 50ul). Ensure the column is not contaminated with peptides and proteins from earlier separations. If possible, do not take proteins to complete dryness, only reduce the volumes. Once dry, many proteins are difficult to solubilise again.
If the sequence is part known, the position of any proline should be indicated in order to reduce overlap when sequencing. If identification of cysteine is required, please inform Alta Bioscience as the sample must be derivatised before sequencing.
Spin columns can be used to extract and concentrate proteins from homogenised gels prior to chemical or enzymic digestion.

QWhat method is used to do the sequencing?

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.

QHow do I send a sample

Most samples can be sent by normal mail in a padded bag, no need to freeze.
Please cut out any gel bands and put them into a vial before sending. ( Gels tend to fall apart in transit and in the freezer ).
A scan or photocopy of the gel or blot can be useful.