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|サンプル脱塩料金、必要な場合 (ABI ProSorb cartridge or a Millipore ZipTip)||8,700円|
- 使用装置：Applied Biosystems 494 Procies High-Throughput Protein Sequencer
Applied Biosystems 494 cLC High Sensitivity Protein Sequencer
- 特異項目：C-末情報提供も可、N-末ブロック蛋白質の解析も可 (別途特別料金)
- N末端エドマン配列決定: 5〜50 pmol (実績としては1 pmol程度の少量精製タンパク質でデータが得られている)
- 内部エドマン配列決定: 5〜100 pmol (試料の分子量に応じて1〜5 μg)
- ゲルサンプル: >50 picomoles (またはクリーンアップが必要な場合、目的のタンパク質バンドを視覚的に識別できる場合)
- PVDF: stained as described below, submitted as a dry membrane.
- 溶液: volume less than 100 microliter, volatile buffer with very little salt content.
- 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.
- ゲル: 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: ご相談ください。
- ニトロセルロース: is not recommended because of lower recovery of peptides.
注: サンプルは1.5 mlポリプロピレン製のマイクロ遠心チューブに入れてください。
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, お問い合わせ下さい.
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.
- 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.
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.
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 (メーカーサイト). 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.
- 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 comfounding 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.