Simple Western™ analysis of lysates (1.5 mg/mL) from parental Jurkat cells, and Jurkat cells engineered to express an scFv-based Anti-CD19 CAR containing a G4S linker (Jurkat/CD19 CAR) using G4S Linker (E7O2V) Rabbit mAb #71645. The virtual lane view (left) shows the target band (as indicated) at a 1:50 dilution of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at a 1:50 dilution of primary antibody in parental Jurkat cells (black line) and Jurkat cells engineered to express an scFv-based Anti-CD19 CAR containing a G4S linker (Jurkat/CD19 CAR) (red line). This antibody is not approved for traditional Western blot. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module. Sample and stacking matrix load times were 6 seconds and 18 seconds, respectively. Baseline points were manually added to the electropherogram.

Flow cytometric analysis of live pan-CD3+ T cells isolated from human PBMCs and engineered to express an scFv-based Anti-CD20 CAR containing a G4S linker, using G4S Linker (E7O2V) Rabbit mAb (right) or concentration-matched Rabbit (DA1E) mAb IgG XP^® Isotype Control #3900 (left). Tag Blue fluorescent protein (TagBFP) is co-expressed with the CAR. Anti-rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4412 was used as a secondary antibody. Data courtesy of Michael Kvorjak, Lohmueller lab (University of Pittsburgh).

Flow cytometric analysis of live Jurkat cells (blue, negative) or Jurkat cells engineered to stably express an scFv-based Anti-CD19 CAR containing a G4S linker (green, positive) using G4S Linker (E7O2V) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP^® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4412 was used as a secondary antibody. Engineered cell line was provided by the Lohmueller Lab, University of Pittsburgh.

Flow cytometric analysis of live Jurkat cells (blue, negative) or Jurkat cells engineered to stably express an scFv-based Anti-CD20 CAR containing a G4S linker (green, positive) using G4S Linker (E7O2V) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP^® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4412 was used as a secondary antibody. Engineered cell line was provided by the Lohmueller Lab, University of Pittsburgh.