Characterization of Phosphopeptides Using a Combination of Immobilized Metal Ion
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Wash the Ni-NTA resin three times each with 30 µL of 100 mM EDTA (pH 8.0) to remove the bound Ni2+ metal.
Wash the column three times each with 30 µL of H2O.
Wash the column three times each with 30 µL of 100 mM acetic acid.
To regenerate the columns with either Fe3+ or Ga3+ metal, wash the column three times each with 30 µL of either 60 mM GaCl3 or 100 mM FeCl3.
To remove any unbound metal ions, wash the column three times each with 30 µL of H2O followed by three times each with 30 µL of 100 mM acetic acid.
Affinity Binding of Phosphopeptides to IMAC Column
- Digest the phosphoprotein (~0.1 µg/µL) with trypsin (protein:enzyme ratio of 20:1 to 100:1 [w/w]) in 50 mM NH4HCO3 buffer (pH 8) for 2 h at 37°C.
- Load 20 µL (~20 µg) of the trypsin-digested phosphorylated protein with 30 µL of 100 mM acetic acid onto the IMAC column. Incubate the column on a slowly rotating wheel for 30 min at 37°C.
Check the column periodically for leakage from the bottom of the CRC. See Troubleshooting.
- Remove unbound peptides from the IMAC column by washing the column with 30 µL of H2O followed by 30 µL of 100 mM acetic acid. Repeat this step three times. The IMAC column (with bound phosphopeptides) can be stored in 100 mM acetic acid for at least 2-3 d at 4°C. Even bound phosphopeptides from a complex mixture such as a cell extract should be stable because endogenous phosphatases are digested by the trypsin and/or elute from the column. To verify that nonspecific peptides are washed off the column or if the unbound peptides are of interest, analyze the washes by MALDI-MS.
- Spot a 0.5-µL aliquot of settled beads with 0.5 µL of MALDI matrix on the MALDI target
Alkaline Phosphatase Digestion of Affinity-Bound Phosphopeptides
- Mix a 5-µL aliquot of the phosphopeptides affinity-bound to the IMAC media (from Step 10) with 5 µL of 50 mM NH4HCO3 (pH 7.8) and 1 unit of calf intestine alkaline phosphatase in a 0.7-mL microcentrifuge tube.
- Incubate the tube on a slowly rotating wheel at 37°C.
- Monitor the time course of the digestion periodically by spotting a 0.5-µL aliquot of the reaction mixture (supernatant and beads) with 0.5 µL of MALDI matrix onto a MALDI target. Take the first aliquot 30 min after beginning Step 13.
Initially, additional aliquots are typically monitored at 30-min intervals. Depending on the protein, incubation times for the digestion can range from 1 h to overnight.
Carboxypeptidase Y Digestion of Affinity-Bound Phosphopeptides
- Transfer a 25-µL aliquot of the phosphopeptides affinity-bound to the IMAC media (from Step 10) into another CRC.
- Wash the column three times each with 30 µL of 50 mM sodium citrate (pH 6).
- Add 40 µL of 50 mM sodium citrate (pH 6) to the column.
- Add 1 µL of carboxypeptidase Y solution to the column.
- Incubate the column on a slowly rotating wheel at 37°C.
- Depending on the protein, the carboxypeptidase Y digests can take from a few minutes to overnight to complete. Thus, the time course of the digest should be monitored initially at 30- to 60-sec intervals. To monitor the time course of the reaction:
- the beads three times each with 50 µL of 100 mM acetic acid.
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