Mouse anti glutamylated tubulin gt335

DRG neurons were fixed in 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% triton-X in PBS for 10 min, and blocked in 15% goat serum/1% BSA/0.1% triton-X in PBS. Primary and secondary antibodies were diluted in block and incubated for 1 hr each at room temperature. The following primary antibodies were used: mouse anti-βIII tubulin (1:800, Sigma), rabbit anti-TUJ1 (1:1000; Covance), chicken anti-GFP (1:500; Aves Labs), mouse anti-glutamylated tubulin GT335 (1:3000; AdipoGen), mouse anti-acetylated tubulin 6-11B-1 (1:1000; Sigma), rat anti-tyrosinated tubulin YL1/2 (1:1000; Abcam), rabbit-anti Tom20 (1:500; Santa Cruz Biotechnology). The following secondary antibodies were used at 1:500: AlexaFluor-488 anti-mouse, anti-rabbit, or anti-chicken, AlexaFluor-568 anti-rabbit, AlexaFluor-647 anti-mouse (Invitrogen), Cy3-conjugated anti-mouse, Cy5-conjugated anti-rat (Jackson ImmunoResearch). Images of fixed cells were acquired on a Nikon Eclipse E800, or when fluorescence was to be quantified, a Zeiss LSM 710 confocal microscope.

Protein lysates were separated by 8% SDS-PAGE and blotted with the following primary antibodies: rabbit anti-TUJ1 (1:2500; Covance), mouse anti-β-actin AC-74 (1:5000; Sigma), mouse anti-glutamylated tubulin GT335 (1:2000; AdipoGen). HRP-conjugated secondary antibodies (1:5000; Jackson ImmunoResearch) and SuperSignal West Dura (Thermo Scientific) were used for chemiluminescent detection.
For assessing levels of glutamylated tubulin in DRG cultures, cells grown directly on poly-d-lysine and laminin coated 24-well plates were lysed in 1X Laemmli sample buffer and an equal volume of each sample was loaded onto the SDS-PAGE gel.