Affinity Purification of Trimethylated Proteins

1.

Prepare the cell lysate with lysate buffer with the proper protease (if it is not the digested peptide)

2.

The crude protein concentration of the lysate should be >5 mg/mL

1.

Wash 50 μL of the anti-acetyl lysine agarose (ICP0606) in a 1.5 mL vial with PBSt three times using a micro-centrifuge at a speed around 1000 rpm and aspirate.

2.

Add the cell lysate (2-10 mg) to the anti-trimethyl lysine agarose beads and incubate in a rotor shaker at room temperature for 60 min.

3.

After incubation, wash the beads with PBSt four times by centrifugation and aspiration.

A) For western blot: simply add 60 μL of the 2x SDS-PAGE loading buffer (without DTT or mercaptoethanol to minimize the release of the IgG) to the beads, vortex and boil for 5 min. Centrifuge the beads at 5000 rpm for 2 min then take the supernatant for analysis following the normal western-blot procedures.

B) For other analysis, elution is needed: elute the bound acetylated proteins from the beads by mixing with 40 μL of glycine-HCl (0.1 M, pH2.5), vortex, then centrifuge at 5000 rpm. Take the supernatant as fraction 1 then repeat twice to obtain fraction 2 and fraction 3. The fractions could be neutralized with saturated Tris if necessary.

1) The simplest method is ELISA: Add 45 uL of 0.1 M Na2CO3 to two wells of the ELISA strip. Add 5 uL of the first IP fraction prepared in B) method to one well and label it as sample; add 5 uL of the residue crude lysate to the other well as control. Incubate at 39oC for 60 min then wash the well with TBST. Add 50 uL of the anti-trimethyl lysine HRP conjugates (0.1 ug/mL in TBST with 1% BSA) then incubate at 37oC for 30 min. Wash the well with TBST four times then add peroxidase substrate TMB to develop color. Successful IP will see blue color in sample well and a significantly lower signal in the control well.

2) Western blot method: Run SDS-PAGE of the IP sample prepared in A) method and perform WB analysis of the sample using 1 ug/mL of the anti-trimethyl lysine HRP. After washing the membrane, directly develop with ECL to see if there are any protein bands with trimethylated lysine residues.

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.