科研技術
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Standard Operation Procedure
Immunoprecipitation of the Acetylated Proteins (or Peptide) with Anti-acetyl Lysine Agarose (Non-column method)
Methods: Immunoprecipitation Procedure 1. Wash 40 mL of the anti-acetyl lysine agarose in a 1.5 mL vial with PBSt three times, using the micro-centrifuge at a speed around 1000 rpm and aspirate. This step is to remove the glycerol. 2. Add the cell lysate (about 2 mg protein/sample) 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 the Agarose if a larger amount of crude protein sample is used). 3. 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 mL 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 2nd antibodies.
For other analysis, elution is needed: Elute the bound acetylated proteins from the Agarose by mixing with 40 mL 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 uL 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 product for blocking or for solution, which may contain acetylated proteins. | |
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Mini-clomun Procedure
1. Pack a mini-column as illustrated in the figure using a piece of glass wool as filter at the bottom of a 1.5 mL pipette tips. Load 100 mL of the anti-acetyl lysine agarose (50 uL bed volume) slurry to the tip, wash the beads with about 3 mLPBSt, using a syringe vaccum to facilitate the washing. This step is to remove the glycerol.
2. Block the mini-column with 1 mL PBSt with 1.5% BSA, let it drop naturally.
3. Pass the cell lysate (about 2 mg protein/sample) or digested crude peptide (pH 6.5-7.5) to the anti-acetyl lysine Agarose column, let it drop naturally. The slower flow rate should be beeter interaction. If the flow rate was too fast, recycle the sample passing several times. The minimum times of sample-column interaction should not be less than 120 minutes at room temperature.
4. After sample passing, wash the column with 5 mL of PBSt, then 2 mL 1M NaCl and final wash with 400 mL water.
5. Elute the bound materials with 120 mL of 0.5 N HCl. Discard the first 20 mL fraction (void), and collect 100 mL 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 product for blocking or for solution, which may contain acetylated proteins.