Quality control is provided by utilizing appropriate internal and external standards as recommended by instrument manufacturers. All analysis utilize the latest analytical software packages.
Our N-terminal protein sequencing uses automated Edman chemistry, carried out on an Applied Biosystems protein sequencer and equipped with an on-line HPLC system. This breaks down a protein/peptide sequentially into its constituent amino acids from the N-terminus of the sample. The amino acid produced is derivatized and separated by RP-HPLC and is visualized by UV detection for each cycle of Edman chemistry. The amino acids are quantified by comparison to a standard mixture. If C-terminal sequencing is required, a mass mapping by MS/MS for research will be conducted for a full characterization of their recombinant protein.
Upon request, we first carry out Edman sequencing and report the first 7 amino acids in the N-terminus of a protein, but some proteins contain signal sequences that may not be correctly processed. Other proteins are truncated as part of the degradation process. While some proteins are N-terminal modified by acetylation, pyroglutamic acid and other blocking groups that will prevent successful N-terminal sequencing. There is not currently an established chemical sequencing method for protein C-terminus.
Protein Sequencing Sample Preparation Guidelines
Proper sample preparation is crucial for optimal protein sequencing results. One important parameter is sample contamination with other proteins because it becomes more difficult to produce useful data as the sequence of the 'target' may be obscured
by the presence of other sequences. Additional considerations such as concentration,volume of sample, and the presence and concentration of detergents, glycerol, buffers and other salts can also affect sequencing result. Prior to sending samples, investigators are recommended to contact our facility to discuss the required analysis. This is necessary to insure that the most efficient and cost-effective analytical methods are employed. Samples are normally analyzed in the order of their receipt, but special arrangements can be made for unstable samples. A sample submission form and guidelines should accompany each set of samples. Consult with our technical support for details.
1. N-terminal Edman sequencing: 5-50 pmol (data has
been obtained with as little as 1 pmol of purified protein).
2. Internal Edman sequencing analysis: 5-100 pmol (1-5μg depending on sample molecular weight).
- Gel samples: >50 picomoles should be supplied for gel samples or if clean-up is required or desired protein band can be visually
Note: Each internal sequence sample must have a suitable blank(negative control).
- N-terminal sequence analysis:
1. PVDF: stained as described below, submitted as a dry membrane.
2. Solution: volume less than 100 microliter, volatile buffer with very little salt content.
- Internal sequence analysis:
- PVDF : stained as described below, dry, maximum practical limitation for sample amount should be approximately 20 lanes on a minigel. Best results are obtained when the sample is concentrated to the fewest number of lanes as possible without overloading.
- Gel: gel samples must be limited to 1-3 lanes from a mini-gel (1mm thick max) and can only be stained with 0.5% Coomassie blue.
- G-250 as described below. Solution: please contact the Bio-Synthesis before submitting sample.
- Nitrocellulose: is not recommended because of lower recovery of peptides.
Note: Please submit all samples in 1.5 ml polypropylene microcentrifuge tubes for efficient handling. Thank you.
- Dry, in solution, as an SDS or native polyacrylamide gel piece,electroblotted to PVDF membrane
CAUTION! Nitrocellulose is not compatible with the Edman chemistry.
- Lyophilized Sample The sample will be reconstituted in 0.05% TFA/50% acetonitrile, 70% formic acid,or 100% TFA for loading, unless specified otherwise. If there will be a solubility problem, please contact us.
- Liquid samples must be >90% pure of a single peptide or protein.
- >10 pmols of pure protein in 30-150 ul of volatile solvents
such as water, acetonitrile, propanol, acetic acid, or formic acid.
- Avoid following reagents:
Buffers and primary amines: Tris buffer is commonly used for protein
purification. Tris and glycine are common in samples recovered from SDS-PAGE.
Glycerol and sucrose: These reagents are often added to buffers
designed for the storage and handling of proteins. These compounds are not volatile
and leave a highly viscous residue.
Nonionic detergents: Triton X-100, Brij, and Tween solutions often
contain aldehydes, oxidants and other contaminates that can inhibit Edman degradation
SDS: Large quantities of SDS can cause instrument malfunction and
may lead to the loss of sample from the filter.
CAUTION! Dialysis tubing is often a source of contaminants and other interfering substances. Avoid dialysis as a last step in sample preparation or use thoroughly cleaned, high-quality tubing. Always dialyzed against a salt counter ion or dilute acid to prevent the protein and contaminates that may be present from sticking to the tubing.
If you are unable to eliminate these materials from your buffers PLEASE DISCUSS WITH THE BSI STAFF BEFORE YOU SUBMIT YOUR SAMPLES.
Please provide SDS gel image when submitting your sample.
Samples purified by SDS-PAGE must be electroblotted onto PVDF membrane and sequenced directly from PVDF membranes. Nitrocellulose membrane is NOT acceptable as it is not resistant to the Edman chemistry. We have recommended protocols for Electroblotting and staining available on our Electroblotting page. In general we recommend
The average sequencing yield from PVDF is approximately 15% instead of the 50-80% expected for solution samples, so a 10 pmols sample on PVDF usually gives 1.5 pmols amino acid peaks. This is due to water vapor that aids PiTC coupling in the Edman chemistry being repelled by the PVDF. Therefore, our preferred stains are old-fashioned Coomassie blue and Ponceau S. or Amido black (silver stains may not be used)
Destained extensively with at least 4 changes of destaining solvent.
Washed with 3-4 changes of ultra-pure water to lower the very high concentrations of Tris, glycine, and other gel and transfer buffers that otherwise will interfere with sequencing.
DO NOT remove all the stain from the bands, they need to be clearly visible for excision as PVDF without protein hinders the flow of chemicals thru the instrument's sample cartridge. This sample cartridges can hold approximately 20 square mm of PVDF membrane. This is roughly equivalent to a slice 1-2 mm high and the width of
three lanes of a mini-gel. We prefer that a non-glycine electroblot buffer be used. Glycine is an amino acid and will contaminate the sample resulting in uninterpretable sequence information for one or two cycles. Please contact us with any questions.
After air drying, the bands or spots of interest should be individually excised
and placed in 1.5 ml Eppendorf tubes for shipment.
- Gels should be stained with Coomassie Blue R-250 or G-250.
- Do not use silver stain.
Protein can be passively eluted from polyacrylamide in an overnight procedure for an additional fee.
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.
Your sample will be loaded into the sequencer cartridge by spotting a pure protein liquid sample onto a Biobrene-saturated glass fiber filter or by placing a small amount of PVDF membrane directly into the sample cartridge. Liquid samples that
contain contaminating or compounding chemicals (see under Liquid Samples) will be loaded onto ProSorb cartridges and washed with 0.2% TFA to remove contaminants before sequencing commences. There is an additional charge for this preparation step.
All reagents and solvents must be of the highest purity available (HPLC grade, sequencing grade and electrophoresis grade reagents) to avoid contaminating substances. Avoid molecular biology grade reagents.
Always wear gloves and work in a clean dust free area. Dust and finger prints are a major source of contaminating amino acids present in sequencing samples.
Avoid drying the sample in glass tubes. This can lead to substantial loss of sample for some proteins. Sample volumes should be less than 150 ul, however with the advent of ABI's Prosorb Sample Preparation Cartridge, sample volumes of up to 750 ul may be sequenced.
The sample should be in a volatile solvent or buffer such as acetic acid, formic acid, trifluoroacetic acid, triethylamine, pyridine, acetonitrile, propanol, water, or ammonium bicarbonate (if lyophilized repeatedly).
A minimum of 10 to 50 pmol of sample should be analyzed. Our Precise sequencer can sequence 1-2 pmol of sample at its highest level of sensitivity. However, it is more practical to sequence larger amounts of protein to be confident of the sequence obtained or to be confident that the N-terminus is blocked if no sequence is obtained.In most cases the amount of sequence able material is underestimated by sample loss,
inaccurate quantitation, or N-terminal blockage during sample preparation. Therefore,be sure to err on the side of too much sample rather than too little!
The sample may contain a small amount of detergent (less than 0.1% SDS). Larger amounts can cause instrument problems.
When submitting electroblotted samples on PVDF for sequencing, always try to have as much protein as you can in as small an area of PVDF as possible. Too much PVDF in the sequencer's reaction cartridge can lead to excessive sequencer background.
One reason why the initial yields are often unexpectedly low, is that the amount of sample present is overestimated by the investigator. The most reliable quantitation method is from amino acid analysis. Lowry, BCA, dye binding assays and absorbance are less accurate methods especially in the low microgram amounts.
Sample Submission and Ordering
We recommend that you send liquid samples in screw cap (with gasket) Eppendorf tubes packed in such a way that are cushioned from the effects of FedEx. If a gasket-equipped tube is not available, use Parafilm to assure that the cap does not pop off in shipping.Keeping the sample cool is at the discretion of the investigator. We can receive packages with wet ice or dry ice but all samples can only be received during normal
Samples for sequencing that are on PVDF membranes do not require being kept cool.We recommend sending dried membranes between two pieces of clean filter paper by FedEx Letter. We discourage you from sending individual slices of PVDF membrane.Also, please include a print (or sketch) of the membrane indicating which band(s)to sequence.
- Before sending a sample, please use
Protein Sequencing Submission Form to alert us for the arrival of protein sample, at same time, a printed copy of same Protein Sequencing Submission form should accompany the sample. This form is available on this website and can be filled out on line. It can be sent electronically and we prefer that it is printed, sign and make sure enclosed along with the sample.
- Ship the sample to:
Attn: Protein Sequencing
612 E. Main Street
Lewisville, TX 75057
800.227.0627 | 972-420-8505
Links and Resources
Order and Quote
Protein Peptide Sequencing Submission
- A Practical Guide to Protein and Peptide Purification. Second Edition. (Paul Matsudaira ed. ), p. 10 - 17, Academic Press, Inc., San Diego, California, 1993.
Edman. P. (1950) Method for Determination of the Amino Acid Sequencing in Peptides.
Acta Chem. Scand., 4. 283-293.
Niall.H.D. (1973) Automated Edman degradation: the protein sequenator. Methods Enzymol.
Matsudaira. P. (1987) Sequence from picomole quantities of proteins electroblotted
onto polyvinylidine difluoride membranes. J. Biol. Chem. 262:10035-10038.
Additional Protein Sequencing Sample Preparation Tips can be found in A Newcomer's
Guide : Preparing Samples for Protein Sequencing at ABI's protein sequencing page.
Fernandez, J., DeMott, M., Atherton, D., and Mische, S.M., (1992) Internal Protein
Sequence Analysis: Enzymatic Digestion for less than 10 Micrograms of Protein Bound
to Polyvinylidene Difluoride or Nitrocellulose Membranes. Anal. Biochem., 201,
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Digestion of Polyvinylidene Difluoride-Bound Proteins for Internal Sequence Analysis.
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"Fernandez, J., Gharahdaghi, F., and Mische, S.M. (1998) Routine identifiaction
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gels or polyvinyl difluoride membranes using matrix assisted laser desorption/ionization-time
of flight-mass spectrometry (MALDI-TOF-MS). Electrophoresis, 19,
Atherton, D., Fernandez, J., DeMott, M., Andrews, L., and Mische, S.M., (1993) Routine
Protein Sequence Analysis Below Ten Picomoles: One Sequencing Facilities Approach,
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Matsudaira, P. (1987) Sequence from Picomole Quantities of Proteins electroblotted
onto Polyvinylidene Difluoride Membranes. J. Biol. Chem., 262,
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Nylon and Hydrophobic Membranes. Anal. Biochem., 173, 18.
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Proteins Transferred to Nitrocellulose after Separation by Sodium Dodecylsulfate-Polyacrylamide
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Schaffner, W., and Weissman, C. (1973) A Rapid, Sensitive and Specific Method for
the Determination of Protein in Dilute Solution. Anal. Biochem., 56,
Simpson, R.J., Moritz, R.L., Nice, E.E., and Grego, B. (1987) A high-Performance
Liquid Chromatography Procedure for Recovering Subnanomole Amounts of Protein from
SDS Gel Electrophoresis for Gas-Phase Sequence Analysis. Eur. J. Biochem., 165,
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from Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications.
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Moos, M., Nguyen, N.Y., and Liu, T.Y., (1988) Reproducible high yield sequencing
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