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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.
Proteins submitted for N-terminal
sequencing
- Sample amount required 20 pmol minimum (ready to sequence), remember
only 50% will yield signal.
- Sample Purity ≥ 85%
Mass to Moles Conversion Table
Mol. Wt. |
1 nmol |
100 pmol |
10 pmol |
1 pmol |
100 fmol |
|
100 kD |
100µg
| 10µg |
1
µg
| 100 ng |
10 ng
|
|
80 kD |
80µg |
8µg |
800 ng |
80 ng |
8 ng |
|
60 kD |
60 µg |
6µg |
600 ng |
60 ng |
6 ng |
|
50 kD |
50 µg |
5µg |
500 ng |
50 ng |
5 ng |
|
40 kD |
40 µg |
4µg |
400 ng |
40 ng |
4 ng |
|
20 kD |
20µg
|
2µg |
200 ng |
20 ng |
2 ng |
|
10 kD |
10µg |
1µg |
100 ng |
10 ng |
1 ng |
|
Electroblotting on PVDF membranes
Count on a loss of 50-75% during
blotting. You will need 20 pmols of desired protein on the PVDF.
IMPORTANT!! Use PVDF membranes only. Nitrocellulose ruins the sequencer.
Common transfer buffers
| 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.
Peptide Synthesis and Purification | Protein Sequence Analysis
Mass Spectrometry | 2D Gel Electrophoresis
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