The GST (glutathione S-transferase) tag is derived from a protein encoded by the parasite Schistosoma japonicum. The 26 kDa GST tag is typically fused to the N-terminus of a recombinant protein. When expressed, GST quickly folds into a stable and highly soluble protein, resulting in greater expression and solubility of recombinant proteins with the GST tag. This can be especially helpful when expressing proteins in bacteria. The GST protein also has a strong affinity for its substrate, GSH (glutathione) and GST-tagged fusion proteins can be detected or purified based on the binding of GST to GSH. However, the GST tag is relatively large compared with other common epitope tags, and it may interfere with some protein functions. In these cases, it may be easily removed by protease cleavage. GST tag antibodies provide another dependable method for the detection and purification of tagged target proteins without a protein-specific antibody or probe. Invitrogen GST tag antibodies are designed to reliably detect GST in various applications.

Applications

Invitrogen GST Tag antibodies are designed to dependably detect GST tag in various applications.

Flow cytometry analysis of GST tag

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 mouse monoclonal antibody (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 Alexa Fluor 488-conjugated rabbit anti-mouse secondary antibody (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-specific mouse monoclonal antibody (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-HRP secondary antibody (Cat. No. 31430) at a dilution of 1:25,000 for at least one hour. Membranes were washed and chemiluminescent detection was performed using Thermo Scientific SuperSignal West Dura Extended Duration Substrate (Cat. No. 34075).

Western blot analysis of GST tag
Immunofluorescence analysis of GST Tag

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 AlexaFluor 647 conjugated GST-tag monoclonal antibody (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.