Anti ha agarose antibody

HA-tagged CDH1 WT and mutants were transfected into HEK293T cells, followed by being immunoprecipitated with monoclonal Anti-HA-Agarose antibody (Sigma, A2095). The purified HA-CDH1 proteins were then incubated with 500 uM of ATPγS (Abcam, ab138911) and 0.5 ug of recombinant human cyclin D1 + CDK4 proteins (Abcam, ab55695) in the kinase reaction buffer (50 mM Tris-HCl, 10 mM MgCl₂, 0.1 mM EDTA, 2 mM DTT, 0.01% Brij 35, pH 7.5) for 30 min at room temperature. Then adding 2 mM of PNBM (Abcam, ab138910) and allowing the alkylating reaction proceed for additional 2 h at room temperature. The reaction was then terminated by adding 5x SDS loading buffer and boiled for 10 min. Samples were then subjected to IB using anti-Thiophosphate ester antibody (Abcam, ab92570).

Western-blotting and immunoprecipitation were performed as described previously [31 (link)]. Briefly, 1 × 10⁶ cells were lysed with lysis buffer [1 × PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and freshly added 100 μg/ml phenylmethanesulfonyl fluoride (PMSF), 10 μ g/ml aprotinin, and 1 mM sodium orthovanadate]. Cell lysates obtained were centrifuged, and protein concentration of the clarified lysates was measured using Easy II Protein Quantitative Kit (BCA). 40 μg of the supernatant protein was separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The blot was blocked with 5% non fat milk, incubated with the indicated antibody, and then incubated with an appropriate peroxidase conjugated secondary antibody. The signal was developed using 4-chloro-1-napthol/3,3-o-diaminobenzidine, and relative photographic density was quantified by a gel documentation and analysis system. GAPDH or HSP70 was used as an internal control to verify basal expression levels and equal protein loading. The ratio of the specific proteins to GAPDH orHSP70 was calculated. 100 μg of the clarified supernatants were immunoprecipitated using anti-FLAG-agarose or anti-HA-agarose antibody (Sigma Chemical Co.). MNAT1 or p53 in the immunoprecipitated complexes was respectively determined by Western-blotting with anti-MNAT1 or anti-p53 antibody.

F. novicida expressing 2xHA- or TEM1-tagged proteins were subcultured to an O.D.₆₀₀ of 1.5 and lysed by sonication in Tris 50mM, NaCl 150mM buffer containing an EDTA-free protease inhibitor cocktail (Roche). HA₂-tagged proteins expressing lysates (equivalent to 4 ml of bacterial culture) or 2% BSA in Tris 50mM, NaCl 50mM as a control were incubated with 10μl of anti-HA agarose antibody (Sigma) slurry for 2h at 4°C. Beads were washed four times with the same buffer and incubated for 3h at 4°C with TEM1-tagged proteins expressing lysates (equivalent to 4 ml of bacterial culture) under mild agitation. Beads were then washed twice with Tris 50mM, NaCl 50mM, NP40 1% and twice with Tris 50mM, NaCl 50mM before being boiled in Laemmli sample buffer (Tris-HCl 50mM, SDS 1%, glycerol 5%, Bromophenol blue 0.0005%) with β-mercaptoethanol (286 μM).