Kcr peptide enrichment

1. Mix anti-crotonyllysine agarose bead (PTM Bio Inc., hangzhou, china) suspension and aliquot 40 μl 50% bead slurry to 0.6 ml tube.

2. Wash beads with 0.5 ml pre-chilled PBS. Spin down beads at 1000 x g for 1 min at 4°C, and remove the supernatants. Repeat twice.

3. Dissolve 2 mg peptides in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% Nonidet P-40, pH 8.0).

4. Remove any possible precipitates in peptide solution by centrifuging at 12,000 x g for 10 min at 4°C;

5. Mix peptide solution with pre-washed antibody conjugated beads. Incubate at 4°C for 4h with gentle shaking.

6. Harvest beads by centrifuging at 1000 x g for 1 min at 4°C.

7. Wash the beads four times with 1 ml of NETN buffer and twice with deionized water.

8. Elute bound peptides with 1% trifluoroacetic acid. Repeat twice and combine all three elutes.

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