Immunoprecipitation of Acetylated Proteins

Wash 40 μL of the anti-acetyl lysine agarose in a 1.5 mL vial with 1 mL PBST three times, using the micro-centrifuge at a speed around 1000 rpm and aspirate. This step is to remove the glycerol.

Wash the anti-acetyl lysine agarose with 1 mL of 0.1 M NaH2PO4 + 1M NaCl twice.

Wash the agarose with 1 mL of PBST.

Add the 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 4oC overnight. (Increase the volume of agarose if a larger amount of crude protein sample is used).

After incubation, wash the agarose beads with PBSt four times, through repeating centrifugation and aspiration.

For western blot: simply add 60 μL of the 2x SDS-PAGE loading buffer (without DTT or Mercaptoethanol) to the beads, vortex and boil for 2 minutes. Centrifuge the beads at 5000 rpm for 2 minutes then use the supernatant for analysis by following the normal western-blot procedure. If you want to detect the acetylated lysine signal, the use of anti-acetyllysine HRP conjugates (ICP0381) is recommended to avoid the IgG interference by the secondary antibodies.

For other analysis, elution is needed: Elute the bound acetylated proteins from the agarose by mixing with 40 μL of 0.5 N HCl, vortex then centrifuge at 5000 rpm for 2 min and take the supernatant as fraction 1. Repeat the procedure twice and obtain fraction 2 and fraction 3, respectively. Pool the three fractions together (120 μL in total). Freeze dry (or concentrate) the pooled fractions for further analysis. Saturated Tris could be used for neutralization if necessary.

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 using a piece of glass wool as a filter at the bottom of a 1.5 mL pipette tip. Load 100 μL of the anti-acetyl lysine agarose (50 μL bed volumes) slurry to the tip, wash the beads with about 3 mL PBST, then with 2 mL 0.1 M NaH2PO4 + 1M NaCl, and then again with 1 mL PBST.

Use a syringe connected to the tips to slowly vacuum to increase the flow rate and facilitate the washing (if necessary). The washing step is to remove the glycerol and non-stable-linked antibodies.

Block the mini-column with 1 mL PBST with 1.5% BSA; let it drop naturally.

Pass the cell lysate (about 2 mg protein/sample) or digested crude peptide (pH 6.5-7.5) through the anti-acetyl lysine agarose column; let it drop naturally. The slower flow rate should be better for interaction. If the flow rate was too fast, recycle the sample passing steps several times. The minimum time of sample-column interaction should not be less than 60 minutes at room temperature.

After sample passing, wash the column with 5 mL of PBSt, then 2 mL 1M NaCl and do a final wash with 400 μL distilled water.

Elute the bound acetylated proteins/peptides with 120 μL of 0.5 N HCl. Discard the first 20 μL fraction (void), and collect 100 μL of the eluting HCl. Freeze dry (or concentrate) the eluting materials for further analysis. Saturated Tris could be used for neutralization if necessary.

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