Rabbit polyclonal anti-actin K84 monomethylated antibody (anti-actin K84me1) was generated using a synthetic peptide with monomethylated lysine84 (77TNWDDMEmKIWHHTFY91) as immunogen (New England Peptide, Boston, USA). The antibody was double affinity purified from rabbit serum (that means, first the sera is run over the unmethylated column to adsorb non-specific antibody, then the flow-through from this column is run over the methylated peptide column) and then lyophilized (New England Peptide, Boston, USA).
Have nitrocellulose membrane ready, draw grid by pencil to indicate the region you are going to blot.
Using narrow-mouth pipet tip, spot 2 μl of samples onto the nitrocellulose membrane at the center of the grid. Minimize the area that the solution penetrates (usually 3-4 mm diam.) by applying it slowly.
Let the membrane dry.
Transfer the membrane to 5 ml Ponceau S Stain solution
Place on an orbital shaker for 15 min at room temperature.
Rinse membrane with ddH2O to achieve desired staining; approximately 1-2 washes of 2 min each will remove the background staining. Record the result by camera.
Wash the membrane 2-3 times with ddH2O for 5 min each to remove Ponceau S.
Block non-specific sites by soaking in 5% Non-Fat milk in TBS-T for 0.5 h at RT.
Incubate with primary antibody (1 μg/ml for purified antibody, 1:1000 dilution for anti-actin K84me1) dissolved in 5% Non-Fat milk in TBS-T for 2 h at RT.
Wash three times with TBS-T (3 × 5 min).
Incubate with secondary antibody conjugated with HRP for 30 min at RT.
Wash three times with TBS-T (3 × 5 min), then once with TBS (5 min).
Incubate with ECL reagent for 5 min and expose X-ray film in the dark room. Try several different lengths of exposure.
Compare the signal from your unknown sample to that of standard and estimate the concentration.
Actin-K84me1 antibody specifically recognizes K84-monomethylated actin (Fig. 1a).
We also verify the anti-actin K84me1 antibody with HA-β-actin K84A/R by immunoblotting (Fig. 1b).
HA-β-actin purification
Prepare the cell lysate using ice cold 0.1% NET Buffer. (4 × 10cm dishes)
Carefully mix EZview Red Anti-HA Affinity Gel beads until completely and uniformly suspended. Add 100 µl of the 50% slurry into a clean 1.5 ml microcentrifuge tube on ice.
Wash beads twice in 0.1% NET Buffer by adding 750 µl of NET 0.1% Buffer to the tube, and centrifuge in a microcentrfuge for approximately 30 s at 6,000 × g. Discard the supernatant.
Whole cell extract was generated by lysing cells in 0.1% NET buffer followed by sonication using a Sonic Dismembrator (10% output, 1 min, with 10 s-on and 20 s-off cycles)
Immediately centrifuge the lysate for 10 min at 14,000 × g in a microcentrifuge at 4ºC to pellet cell debris.
Carefully remove the clear lysate supernatant from step 5 with a 1 ml micropipette and transfer into the tube of equilibrated EZview Red Anti-HA Affinity Gel beads from step 3. Incubate with thorough, gentle mixing for 3 h at 4°C.
Centrifuge in a microcentrifuge for 30 s at 6,000 × g. Aspirate supernatant carefully.
Wash the bead pellet by adding 1 ml of TBS. Incubate with thorough, gentle mixing at 4ºC for 5 min. Centrifuge in a microcentrifuge for 30 s at 6,000 × g. Aspirate supernatant carefully and set the tube with the bead pellet on ice.
Repeat washes two more times as in step 8.
Elution of the HA-fusion protein with HA peptide. The HA-tagged protein bound to the resin may be eluted with HA peptide. Add the desired volume of a freshly prepared 100 µg/ml solution of HA peptide in 50 mM pH 7.4 Tris-HCl buffer. Incubate the affinity gel sample for 10 min and recover the supernatant after pelleting the affinity gel by centrifugation.
HA-β-actin precipitation:
Add 4 fold volume of –20ºC acetone to ice-cold sample solution,
Mix well and keep at –20ºC for at least 30 min.
Spin in a refrigerated microfuge at top speed for 30 min.
Remove supernatant and air-dry the pellet.
Protease digestion:
Resuspend the pellet in 8 M urea, 100 mM Tris, pH 8.5 (for 0.5 ml urea buffer, mix 240 mg urea, 100 μl 500 mM Tris pH8.5, and 220 μl H2O) and sonicate it for 10 min.
Add TCEP to final concentration of 5 mM and incubate at RT for 20 min
Add iodoacetamide to 10 mM and incubate at RT for 15 min in the dark.
Dilute to 2 M urea with 100 mM Tris-HCl, pH 8.5
Add methylamine to 20 mM to reduce carbamylation
Add Asp-N (according to 1:50 enzyme : substrate), incubate overnight at 37 ºC in the dark
Add EDTA to 0.5 mM to inactivate Asp-N
Add DTT to 5 mM, CaCl2 to 8.5 mM and Arg-C (according to 1:50 enzyme : substrate), incubate for 8 hours at 37 ºC in the dark
Quench with 5% Formic acid
Desalting:
Load samples onto a precolumn (250 μm ID) with 2 cm C18 resin (10 μm) under flow rate of about 1 μl/min.
Wash with 0.1% Formic acid for 10 min.
Elute with 20 μl 80% ACN, 0.1% Formic acid.
Dry with Centrivap.
Resuspend with the NET buffer (without NP-40).
Actin K84me1 peptide enrichment
Take 20 μl of protein A beads slurry for one sample, spin at 6,000 for 1 min, at 4ºC. Discard the supernatant.
Wash beads twice in NET Buffer (without NP-40) by adding 750 µl of NET Buffer to the tube, and centrifuge in a microcentrfuge for approximately 30 s at 6,000 × g. Discard the supernatant.
Add peptides to the beads. Then add proper NET buffer to this tube up to 500 µl and incubate in 4ºC rotator for 60 min.
Spin at 10,000 × g for 3 minutes and transfer the supernatant to a new microcentrifuge tube.
Add 2 µg of purified Rabbit IgG control antibody or 2 µg anti-K84me1 antibody to the pre clean cell lysate. Incubate for 2 h hour in the 4ºC rotator.
Add 40 µl of the protein A beads to a new microcentrifuge tube and wash it 3 times with NET buffer (without NP-40).
Add the lysate and antibody mixture to the washed beads and incubate for 1 h at 4ºC with gentle agitation.
Spin in centrifuge at 6,000 × g at 4ºC for 1 min.
Discard supernatant and wash the beads in 1 × TBS buffer (add 1mM PMSF before used) 5 times (each time centrifuging at 4ºC and removing the supernatant). Each time agitate the tube in the e 4ºC rotator for 5 min.
Then spin down the beads at 6,000 × g at 4ºC for 2 min. Remove the supernatant carefully and completely.
Add 70 µl elution buffer (0.1 M glycine, pH 2.5) to each sample and control resin.
Incubate the samples and controls with gentle shaking for 10 min at room temperature. Do not leave the resin in this buffer more than 20 min.
Centrifuge the resin for 1 min at 8,200 × g. Transfer the supernatants to fresh test tubes containing 7 μl 1 M Tris HCl, pH 8.0. Be careful not to transfer any resin.
Desalting:
Load samples onto a precolumn (250 μm ID) with 2 cm C18 resin (10 μm) under flow rate of about 1 μl/min.
Wash with 0.1% Formic acid for 10 min.
Elute with 20 μl 80% ACN, 0.1% Formic acid.
Dry with Centrivap.
Resuspend with 0.1% Formic acid.
On-line LC-MS analysis:
EASY-nLC 1000 with Q Extactive can automat analyzes samples from the sample bottle.
Analytical column: 75 μm ID with a pulled tip less than 5 μm, packed with 12 cm C18 (3 μm)
Full scan: microscans 1; Resolution 70,000; AGC target 3e6; Maximum IT 60 ms; Number of scan ranges 1; Scan range 400 to 2000 m/z.
dd-MS2: microscans 1; Resolution 17,500; AGC target 1e5; Maximum IT 120 ms; loop count 10; MSX count 1; TopN 10; Isolation window 2.0 m/z; Fixed first mass 100.0 m/z; NCE 27.0; under fill ratio 1.0%; Intensity threshold 8.3e3; Charge exclusion unassigned, 1, 5-8, >8; Dynamic exclusion 150.0 s; if idle pick others
For peptide identification, the MS2 spectra were searched against an EPI-IPI human database (forward+ reversed sequences) using Prolucid with 50 p.p.m. mass accuracy for both precursor and fragment ions and considering carbamidomethylation on Cysteine as a fixed modification and mono-, di- or tri-methylation atLys as differential modifications12. Search results were filtered using DTASelect 2.0with 10 p.p.m. mass accuracy for precursor mass and a 1% FDR cutoff at the spectral level13. The b-actin K84me1 spectra presented in the figures were annotated using pLabel, requiring 20 p.p.m. accuracy for fragment ions14. The data files have been uploaded to ttp://www.peptideatlas.org/ with the access number: PASS00132 and password: WW685h.