Ab170931

The cells were washed twice with Ca/Mg-free PBS and then treated with a microtubule lysing buffer consisting of 100 mM PIPES, 5 mM MgCl₂, 1 mM EGTA, 30% glycerol, 0.1% IGEPAL, 0.1% Tween-20, 0.1% Triton X-100, 0.1% beta-mercaptoethanol, 1 mM ATP, 0.1 mM GTP and a complete protease inhibitor cocktail (Roche); the recipe is according to Cytoskeleton, Inc. (USA). The lysate was homogenized by Retsch Mixer Mill at 25 Hz for 2 min, and incubated for 30 min at 35 °C. The obtained cell lysates were clarified by centrifugation at 21000 x g for 40 min at 35 °C. The protein concentration in lysates was determined using the Pierce BCA Protein Kit. Proteins were separated by 12% SDS-PAGE and transferred onto the PVDF membrane by Trans-Blot Semi-Dry Transfer system (Bio-Rad, Inc., USA).
To determine the presence of beta-tubulin isotypes Abcam mono- and polyclonal antibodies (anti-beta I Tubulin (ab11312), anti-Tubb2A (ab170931) and anti-beta III Tubulin (ab52901) were used. After the chemiluminescence reaction, the PVDF membranes were stained with Coomassie brilliant blue R250 to measure the total protein amount. The tubulin signal intensity was normalized against total protein intensities obtained from Coomassie staining. Quantification was performed by ImageJ software.