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General sample handling
To minimize protein losses from adsorption to walls of tubes, use polypropylene tubes
or siliconized glass tubes. Avoid polystyrene (clear plastic) and untreated glass.
Use freshly prepared, high purity reagents and water. Contaminants from buffers,
detergents, urea, guanidine can prevent a good sequencing result.
Free acrylamide can react with the amino groups on your protein during polyacrylamide
gel electrophoresis which reduces the chance for good sequencing data. Use completely
polymerized gels by either using precasted gels (InVitrogen) or by pouring the separation
gel the day before you plan on running the gel.
Always run standard proteins or markers and a blank on the same gel. Protein quantity
can be estimated by running the appropriate molecular weight markers in differing
concentrations on the same gel as the sample.
Protein Purity and Concentration
The purity of the protein should be 85% or higher. Contaminating peptides or proteins
interfere with the Edman chemistry and contribute to a higher noise level. The lower the
amount of protein, the higher the purity is required.
Proteins submitted for N-terminal
sequencing
- Sample amount required 10 pmol minimum (ready to sequence), remember
only 50% will yield signal.
Mass to Moles Conversion Table
Mol. Wt. |
1 nmol |
100 pmol |
10 pmol |
1 pmol |
100 fmol |
100 kD |
100 µg |
10 µg |
1 mg |
100 ng |
10 ng |
80 kD |
80 µg |
8 µg |
800 ng |
80 ng |
8 ng |
60 kD |
60 µg |
6 µg |
600 ng |
60 ng |
6 ng |
50 kD |
50 µg |
5 µg |
500 ng |
50 ng |
5 ng |
40 kD |
40 µg |
4 µg |
400 ng |
40 ng |
4 ng |
20 kD |
20 µg |
2 µg |
200 ng |
20 ng |
2 ng |
10 kD |
10 µg |
1 µg |
100 ng |
10 ng |
1 ng |
- Proteins blotted on PVDF This is the ideal preparation for sequencing
(see section below).
Proteins in solution Submit
sample volumes between 100 and 1000 microliters.
Please be very explicit on last step in sample preparation. All buffers, solvent,
detergents and their concentrations must be specified. If the sample has been stored for a
period of months indicate also.
Electroblotting on PVDF membranes
Please determine how much protein you have on an analytical gel before you blot. Obtain
standards of known concentrations from the facility. Then consult with us to see if you
have enough protein before you blot you protein to PVDF. Count on a loss of 50-75% during
blotting. You will need 10 pmols of protein on the PVDF.
IMPORTANT!! Use PVDF membranes only. Nitrocellulose ruins the sequencer.
Pre-Wet PVDF with 100% methanol for 30 sec followed by equilibration in transfer
buffer for 5-15 min.
| CAPS: |
10 mM CAPS, 10% methanol, pH 11.0. Degas before use. |
| Tris-Glycine: |
25 mM Tris, 192 mM Glycine, 20% methanol, pH 8.3. |
- CAPS buffer is preferred for sequence analysis (no glycine
contamination). If Tris-Glycine is used the membrane needs to be washed thoroughly with
dd- water following transfer and staining.
User bulletin on electroblotting and references are available.
Proteins
submitted for Internal Sequencing Analysis
 Procedure:
In-solution (purified proteins) or in-gel or digest, analysis of the peptide
mixture by mass spectrometry (peptide mass fingerprinting) or isolation of
peptides by capillary HPLC and subsequent sequence- and mass spectral analysis.
Purified
proteins: Must be submitted in volatile buffers or solvents (trifluoroacetic
acid, formic acid, acetic acid, ammonium bicarbonate, acetonitrile or
triethylamine). In case the protein is not soluble in one of these volatile
buffers, concentrate or precipitate the protein and run it on SDS-PAGE .
Non-pure
proteins should be run on SDS-PAGE, individual bands will be excised for in-gel
digestion.
- In-gel digestion: Polymerize the gel overnight and use 5.0% acetic
acid/20% methanol in the Coomassie Blue G-250 staining solution. Destain the
gel with 30% methanol; the gel does not have to be completely destained.
Important: maximize the protein/gel ratio: concentrate your
proteins, run it in as few lanes as possible without sacrificing resolution.
Peptide Synthesis and Purification | Protein Sequence Analysis
Mass Spectrometry | 2D Gel Electrophoresis
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