GST Tag Antibodies

Flow cytometry analysis of GST tag on HEK-293 cells transiently expressing GST-GAD65. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton X-100 for 20 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with GST Tag Monoclonal Antibody (GST 3-4C) (Cat. No. 136700, red histogram) or with mouse isotype control (pink histogram) at 1-3 µg/million cells in 2.5% BSA. After incubation at room temperature for 2–3 hours, the cells were labeled with Rabbit anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Cat. No. A11059) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Applied Biosystems Attune NxT Flow Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Western blot analysis of GST-Abl SH2 domain (Lane 1, 35kDa) and GST-Grb2 SH2 domain (Lane 2, 38kDa). Analysis was performed by loading 2 µg of each purified fusion protein onto a 4–20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/0.1%TBST for at least 1 hour. Membranes were then probed with a GST Tag Monoclonal Antibody (8-326) (Cat. No. MA4-004) at a dilution of 1:1,000 overnight at 4°C on a rocking platform. Membranes were then washed in TBS/0.1%Tween 20 and probed with a Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (Cat. No. 31430) at a dilution of 1:25,000 for at least one hour. Membranes were washed and chemiluminescent detection was performed using SuperSignal West Dura Extended Duration Substrate (Cat. No. 34075).

Immunofluorescent analysis of HeLa cells. Cells were transfected with a construct containing a GST epitope tag. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature and blocked with 3% Blocker BSA (Cat. No. 37525) for 15 minutes at room temperature. Cells were stained with GST Tag Monoclonal Antibody (8-326), Alexa Fluor 647 (Cat. No. MA4-004-A647) at a dilution of 1:25 for 1 hour at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye (Cat. No. 62249). Images were taken on a Thermo Scientific ToxInsight at 20X magnification.