Western blot was performed using Anti-GST Tag Polyclonal Antibody (Product # PA1-982A) by loading whole cell extracts of untransfected and transiently transfected HEK-293E lysates: untransfected, 50 µg (Lane 1), empty vector control, 50 µg (Lane 2), H3-GST, 50 µg (Lane 3), H3-GST, 25 µg (Lane 4), H3-GST, 12.5 µg (Lane 5), H3-GST, 6.25 µg (Lane 6), GST-p65, 50 µg (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). A 42 kDa band of H3-GST and a 87 kDa band of GST-p65 were observed in transfected lysates on probing with primary antibody (1:2000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1500 (Product # A44241). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
Western blot analysis of GST was performed by loading 25 µg of 293t cells transfected with GST-MART1 (lane 1), HepG2 cells (lane 2) and 293t cells transfected with 12Tag (lane 3) onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a GST Epitope Tag polyclonal antibody (Product # PA1-982A) at a dilution of 1:5000 (GST and 12Tag) and 1:1000 (HepG2) overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). The image shows the target bands on GST-MART1 (39 kDa), HepG2 (26 kDa) and 12Tag (45 kDa), which are consistent with predicted molecular weights for each target. Exposure times were 2 min (lane 1), 1 min (lane 2) and 30 sec (lane 3).
Supplier Page from Thermo Fisher Scientific for GST Tag Antibody