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The following may be helpful in the preparation of samples for submission.
Amino acids are sequentially cleaved from the N-terminus of proteins via Edman Degradation. In this process the N-terminal amino acid is reacted with PITC (phenylisothiocyanate) to form a the PTC-protein with an intermediate anilinothiazolinone forming when contacting trifluoroacetic acid. The intermediate is cleaved and converted to the phenylthiohydantoin (PTH-Amino Acid) form and subsequently separated by HPLC, compared against a standard and identified by the sequencer software.
Any reagent that may potentially interfere with this chemistry should be avoided. These include:
- Non-volatile buffers
- Non-volatile amines
- Free amino acids
- Detergents and salts
Sample loss of small amounts of protein is often caused by adsorption to purification apparatus. To avoid this problem try eliminating as many handling steps as possible.
- Avoid dialysis by applying sample from gel filtration or ion exchange chromatography directly to a reverse phase HPLC separation set up.
- Use polyethylene or polypropylene micro centrifuge tubes keeping protein mass to plastic area as high as possible.
- Avoid drying or lyophilizing samples completely.
Many proteins may be blocked at the N-terminus either by natural processes or by modification during manipulation and purification. Modifications include:
- Formyl groups
- Acetyl groups
- Pyroglutamic acid residues
Modification to the N-terminus can occur via the following:
- Handling steps at elevated pH (>9.0)
- Inferior grade reagents and water (use the highest quality available)
- Due to an exposure to elevated temperatures if a glutamine is the N-terminal residue
- Protease inhibitors may react with amino groups
- Use of formic acid (e.g. in CNBr cleavage) may formylate the N-terminus- use 50-70% TFA as an alternative
- Not deionizing reagents such as urea - urea can be deionized on an ion exchange resin (e.g. Amberlite) prior to use
If Your Sample Is N-Terminally Blocked
If the sample is blocked, either removal of the blocking group or fragmentation (e.g. enzymatic or chemical digestion) is required to obtain sequence data, with fragmentation being the preferred option.
Chemical and Enzymatic Digestion
Large peptide fragments (from methionine containing proteins) can be generated via chemical digestion with cyanogen bromide. Smaller peptides can be obtained from enzymatic digestion with trypsin to endoproteinase Lys-C. Digestion of a salt free (>200picomole) sample, subsequent narrow bore HPLC separation and collection of resulting fragments is offered as a service of the core facility.
Tips for SDS Gel Electrophoresis Prior to Sequencing
- Use ultra-pure reagents.
- Make up stock acrylamide solution fresh every 2 to 3 weeks.
- De-ionize the acrylamide stock solution with ion exchange resin and store it in a brown glass container in a cool place.
- Polymerize gel overnight to minimize any amino-reactive free acrylamide.
- Pre-run the gel (5-10 minutes) before loading your sample.
- Denature sample for 0.5-1hr at 65C - avoid boiling sample.
PVDF Types for Transferring Sequencing Samples from SDS Gels
Weak binding PVDF-suitable for proteins from 5kD up to <100kD
Immobilon-P membranes/Millipore Corp.
Strong binders-more suitable for small polpeptides (<5kD)
- Perkin Elmer/Applied Biosystems' "ProBlott"
- BioRad's "Transblot"
- Millipore's "Immobilon P(SQ)"
Staining PVDF for Sequencing
Samples transferred to PVDF may be stained with Coomassie Blue, Poceau S or Amido black 10B
Information Required for Sequencing
Protein should be at least 80% pure. Purity below this point makes it difficult to identify which residue resides with which protein. A HPLC clean up of your sample is recommended. Please contact the facility operating manager for further information on core services of this nature.
A minimum of 100 picomoles of material is requested. Sequence can be obtained from less material (we have obtained sequence from samples with a starting amount of 10 picomoles), however, for lesser amounts, yield and quality of sequence decreases as sample starting material decreases. It is suggested that 3-5x as much sample as needed is prepared. For samples bound to PVDF please check with the facility operating manager prior to transferring your sample.
Approximate molecular weight.
Cysteine containing and modifying procedure used.
Is the protein glycosylated and to what extent? (if known)
What buffer the sample is in, if any.