Immunoprecipitation of Acetylated Proteins

Immunoprecipitation Procedure
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.
Recovery of the Bound Acetylated Proteins
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.
Notedo not use milk or milk products for blocking or for the solution, which may contain acetylated proteins.
Mini-Column Procedure
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