Mouse anti gst antibody

Protein samples were separated by 10% SDS-PAGE, followed by transferring to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Cat ISEQ00010). After blocking with 5% milk, the membranes were incubated with the primary antibodies mouse anti-Flag antibody (1:3000) (Proteintech, Cat 66008-3-Ig), mouse anti-Myc antibody (1:3000) (Proteintech, Cat 60003-2-Ig), mouse anti-Rab18 antibody (1:500) (Santa Cruz Biotechnology, Cat sc-393168), mouse anti-GST antibody (1:2000) (Abcam, Cat ab19256), or rabbit anti-actin antibody (1:5000) (Abcam, Cat ab8227) at 4°C for 12 h. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Proteintech, Cat SA00001-1) secondary antibody (1:5000) for 2 h. Finally, the signal was detected using an enhanced chemiluminescence (ECL) western blot analysis system. β-actin served as an internal control protein.

Hexa-His tagged or GST tagged chIL-2, which was freshly purified from E. coli or Sf9 cells was treated with 5× protein loading buffer without reducing reagent (125 mM Tris·HCl, pH 6.8, 5% (w/v) SDS, 0.2% (w/v) bromophenol blue, 25% (v/v) glycerol) and boiled. SDS-PAGE (15% gel percentage) was performed to identify purified chIL-2 followed by western blotting using mouse anti-chIL-2 IgG antibody (1:5000 dilution, Bio-rad, Hercules, CA, USA). The GST tag was detected using the mouse anti-GST antibody (1:5000, Abcam, Cambridge, MA, USA). The secondary antibody used was goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) (1:5000, Abcam). The band position detected by anti-chIL-2 antibody was calculated using ImageJ software (V1.52p, NIH, Bethesda, MD, USA) [37 (link)].