Immunoprecipitation of Acetylated Proteins

Prepare beads: Gently resuspend the anti-acetyl lysine agarose beads and transfer the desired amount (typically 20–50 µl of bead slurry per sample) to a 1.5 mL vial.

Wash beads: 

1. Wash the beads with 1 mL PBSt three times, Centrifuge at low speed 1000 rpm for one minute between washes and aspirate the supernatant. This removes the storage buffer and glycerol.
2. Wash the beads with 1 mL of 0.1 M NaH₂PO₄ + 1M NaCl twice.
3. Finally, wash the beads with 1 mL of PBSt.

Incubate lysate: Add the prepared cell lysate (about 2 mg protein/50 µL beads) or digested crude peptide to the anti-acetyl lysine agarose beads and incubate on a rotor shaker at room temperature, for 120 minutes or 4^oC overnight. (Increase the volume of the Agarose if a larger amount of crude protein sample is used).

After incubation, wash the agarose beads with PBSt four times, through repeated centrifugation and aspiration. Finally wash with PBS to remove the Tween-20.

For western blot:

1. Add 60 µL of 2× SDS-PAGE loading buffer (without DTT or β-mercaptoethanol) to the beads.
2. Vortex briefly to mix and then boil for 2 minutes.
3. Centrifuge the beads at 5,000 rpm for 2 minutes.
4. Collect the supernatant and use it for SDS-PAGE and Western blotting according to your standard protocol.

Note: To detect acetylated lysine, it is recommended to use anti-acetyl lysine HRP conjugates (ICP0381). This avoids interference from IgG secondary antibodies.

For Downstream Analysis:

1. Add 60 µL of 0.5 N HCl to the beads and vortex briefly.
2. Centrifuge at 5,000 rpm for 2 minutes and collect the supernatant as Fraction 1.
3. Repeat the elution two more times to obtain Fractions 2 and 3.
4. Pool all three fractions(total volume ~180 µL).
5. Freeze-dry or concentrate the pooled fractions for further downstream analysis.

Note: do not use milk or milk products for blocking or for the solution, which may contain acetylated proteins.

Pack a mini-column (as illustrated in the figure above):

Insert a small piece of glass wool at the bottom of a 5 mL pipette tip to act as a filter. Add 100 µL of anti-acetyl-lysine agarose slurry(equivalent to 50 µL bead volume) into the tip. Wash the beads sequentially as follows:

• 3 mL PBSt (PBS + 0.1% Tween-20);
• 2 mL 0.1 M NaH₂PO₄ + 1 M NaCl; and
• 1 mL PBSt (final wash)

Apply vacuum (optional):
If necessary, connect the pipette tip column to a syringe and gently create vacuum to increase the flow rate and facilitate washing. The washing step removes glycerol and loosely bound (non-covalently linked) antibodies.

Block the column:
Add 1 mL PBSt containing 1.5% BSA to the column and allow it to flow through naturally by gravity.

Load the sample:
Apply the cell lysate (about 2 mg total protein per sample) or digested crude peptide (pH 6.5–7.5) to the column. Let the sample flow through naturally. A slower flow rate provides better binding. If the flow rate is too fast, reload the sample several times. The total contact time between sample and column should be at least 60 minutes at room temperature.

Wash the column: Wash sequentially with:

• 5 mL PBSt,
• 2 mL 1 M NaCl, and
• 400 µL distilled water(final rinse).

Note: do not use milk or milk products for blocking or for solution, which may contain acetylated proteins.