Rabbit anti halotag antibody

Halo tagged recombinant proteins were expressed by in vitro transcription/translation system, and then subjected for Western Blot. Briefly, a total of 3 µl protein lysate was loaded onto 12% SDS-PAGE, then electrophoretically transferred to PVDF membrane (GE). The protein lysate with the HaloTag protein only (31 kDa) was loading as control. After blocking, the membranes were blotted with rabbit anti-HaloTag antibody (Promega), followed by incubation with horseradish peroxidase-conjugated secondary antibody (Proteintech). The protein signals were detected by ECL reagent (GE), and photographed using Alpha Innotech Fluor Chem FC2 chemiluminescence image analysis system (Alpha Innotech).

The cDNAs for 14 autoantigens were cloned into the Flexi vector (Promega, USA) with a HaloTag at N-terminus. The Halo tagged recombinant proteins were expressed by in vitro transcription/translation system (Promega). The HaloTag protein was also produced as negative protein. The proteins were detected by Western blot using rabbit anti-HaloTag antibody (Promega).

Cells on coverslips were fixed in 4% paraformaldehyde in PBS for 10 min and then washed three times briefly in PBS, permeabilized for 20 min at room temperature with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in PBS, washed in PBS 3 times, and then blocked with 3% goat serum (Thermofisher Scientific, Waltham, MA, USA) in PBS for 20 min. Cells were then incubated for 1 h at room temperature with a 1:200 dilution of the rabbit anti-Halotag antibody (Promega, Madison, WI, USA). Cells were washed three times with PBS, and then incubated for 1 h at room temperature with a 1:200 dilution of Alexa Fluor 546 goat anti-rabbit antibody and a 1/1000 dilution of DAPI. Cells were again washed in PBS 3 times, then the coverslips were mounted onto a slide glass with a Pro-long Gold Antifade mounting medium (Thermofisher Scientific, Waltham, MA, USA). Images were taken with an Olympus Fluoview FV-10i confocal microscope (Olympus, Okaya, Japan). The lengths of the microvilli with incorporated GFP-espin1 were measured with ImageJ software (NIH, Bethesda, MD, USA) and statistical significance assessed by Student’s t-test.