Questions
Why should I sequence my protein?
Why use Enzyme Research Proteomics?
What kind of sample(s) can you sequence?
How do I send the samples?
How much sample is required?
How pure does my protein have to be?
I am a liquid. What volume should I send?
I am sending a blot. What membrane should I use?
What transfer buffer should I use for blotting?
What stain should I use for staining?
Are there any other considerations for sample preparation?
How long a sequence do I need?
My sample contains Cysteine or is post translationally modified?
Why didn’t my sample sequence?
I have a question?
Why should I sequence my protein?
The protein sequence provides invaluable information to researchers. It’s main uses include:- - Helping in confirming the identity of a protein.
- Provide information to assist in the design of oligonucleotide/PCR primers.
- Provide data for the design of synthetic peptides for antibody generation.
Why use Enzyme Research Proteomics?
ERP’s has extensive experience in protein sequencing. The protein sequencing facility has three automated state of the art Applied Biosystems instruments (477A, procise 491 and procise 494). Turnaround is rapid with very little down time.
What kind of sample(s) can you sequence?
We routinely sequence proteins in solution and on PVDF blots. For peptides below 5kDa we recommend submitting samples in solution since they tend to be difficult to resolve by SDS-PAGE and/or blot.
How do I send the samples?
Since we are looking at sequence and not activity most samples can be sent by mail in a padded envelope. No special shipping requirements are necessary. However for precious samples we recommend sending samples by courier.
A scan or photocopy of the gel to accompany the sample may be helpful.
How much sample is required?
Sequencing is often limited by an inability to obtain sufficient amounts of adequately purified protein. The minimum quantity of material required for sequencing is between 10 and 100 pmols. The amount of sequenceable protein may be considerably less than the amount of protein so the more material supplied the better. We will gladly attempt to sequence lower amounts if you are unable to obtain larger quantities.
For a typical blot
- Band 1 would give a nice sequence. It is homogenoues and is approximately 40 pmol.
- Band 2 though not homogeneous is >85% pure and could be sequenced.
- Band 3-5 are not present in sufficient quantity.
- Band 6 is not homogeneous. Although not sufficiently resolved a sequence could be attempted.
- Band 7-10 would need several lanes to be combined in order to sequence. It would be at the lower limit of detection.
How pure does my protein have to be?
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 Liquid samples should be greater than 85% pure. Ideally samples should contain just one peptide protein. Samples submitted on PVDF (blotted from SDS-PAGE gels) should have well separated bands to minimize contamination.
I am a liquid. What volume should I send?
A volume of between 30-150 ul (approximately 0.1 mg/ml) would be ideal for absorption onto PVDF membrane using a Prosorb catridge.
I am sending a blot. What membrane should I use?
The ideal medium is a Problot PVDF membrane. PVDF membranes can be supplied on request (info@enzymeproteomics.com). Other PVDF membranes that work well include the Westran membranes from BioRad and Immobilon Psq from Millipore. These membranes are favoured due to their high protein adsorption and 0.2 um pore size. The sample should be as concentrated as possible on the PVDF membrane (e.g. 1 µg/lane). Several bands from replicate lanes can be used. The sequencer can work with 2 to 3 bands worth of blot, (or about 6mm x 8mm in area). Please do NOT send samples on nitrocellulose blots. Nitrocellulose degrades in the sequencer, gums up the tubing and may result in time consuming and costly repairs.
What transfer buffer should I use for blotting?
Ideally we would recommend staying clear of primary amines (eg. Glycine, Tris) as these will interfere with sequencing. Although primary amines can be removed through extensive washing with water a better option would be to avoid using them. Consequently we recommend using CAPS buffer (10 mM CAPS, 10% methanol, pH 11.0). .
What stain should I use for staining?
The bands should be stained with Coomassie Blue, Ponceau S or Amido Black. After staining and destaining the blotted membrane should be rinsed thoroughly with deionized water to remove contaminants. Gold or Silver stains should NOT be used since both appear to block the protein sequencing. Antibodies should NOT be use since these will end up being sequenced and therefore mask the protein of interest. The whole membrane may be submitted with the bands clearly marked. Alternatively the bands of interest may be cut out from the membrane and submitted for analysis.
Are there any other considerations for sample preparation?
Buffers, SDS, salts, amino acids, primary amines, and other contaminants must be removed from your sample. These contaminants affect Edman degradation reaction on the instrument, contaminate the instrument or affect PTH amino acid detection.
How long a sequence do I need?
For most applications long sequence reads are unnecessary. An N-terminal sequence of 10-20 amino acids is usually sufficient for peptide/protein verification. Sequences of 15 amino acids are sometimes required for the construction of DNA probes.Longer sequence reads are possible. However since signal strength reduces upon consecutive cycles there is a maximum limit of around 40 residues.
My sample contains Cysteine or is post translationally modified?
Cysteine will give a blank result unless it is reduced and alkylated prior to sequencing. Acrylamide is the preferred alkylating agent.Post translationally modified residues (eg. glycosylated or phosphorylated) will also give a blank cycle upon sequencing.
Why didn’t my sample sequence?
N-terminal protein sequencing is dependent on the nature and quantity of the sample and hence there is no guarantee that every sample will sequence.Studies on the amino terminal of soluble proteins from a variety of eukaryotic cells suggest that as many as 50% of the alpha-NH2 groups are blocked. The blockage being predominantly alpha-N-acetylation. Where N-terminal blockage occurs sequencing is impossible since there is no free amino terminal available for coupling. In cases where N-terminal blockage occurs internal sequencing is recommended. In situations where the band submitted for analysis is faint and the quantity low, it is sometimes difficult to say whether the fault is due to insufficient material or that the proteins is N-terminally blocked.
I have a question?
If you have a question however trivial that is not answered above please email info@enzymeproteomics.com for response.
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