Evaluation and Validation of Commercially Available Dopamine Transporter Antibodies

Western blots depicting the remaining anti-DAT antibodies with N-terminal immunogens. All western blots were performed using mouse striatum samples from DAT-knock-outs (DKO), and wild-type (WT) mice. Total cell lysates were also obtained and utilized from HEK293 and DAT-expressing HEK293 cells. Molecular ladder is indicated beside each figure, with detection of the dopamine transporter (DAT) indicated by a black arrow. Western blots include: (A) anti-DAT antibody PT-22524-1-AP depicting a strong signal with nonspecific binding on a homemade 10% Tris-Glycin acrylamide gel using total cell lysate, (B) anti-DAT antibody PT-22524-1-AP depicting strong nonspecific signal on a homemade 10% Tris-Glycin acrylamide gel using a mouse striatum sample, (C) anti-DAT antibody D6944 depicting a strong DAT signal with some nonspecific binding on a homemade 10% Tris-Glycin acrylamide gel using mouse striatum sample, and (D) anti-DAT antibody MA5-24795 depicting a strong DAT signal with minor nonspecific binding on a Novex 10% Tris-Glycine gel using mouse striatum sample.

Western blots depicting anti-DAT antibodies with C-terminal immunogens. All western blots were performed using mouse striatum samples for wild-type (WT) and DAT-knock-outs (DKO) mice. Molecular ladder is indicated beside each figure, with detection of the dopamine transporter (DAT) indicated by a black arrow. Western blots include: (A) anti-DAT antibody 431-DATC depicting a moderate signal with some nonspecific binding on a homemade 10% Tris-Glycin acrylamide gel using mouse striatum sample, (B) 600-401-C75 depicting no signal run on a Novex 4–20% Tris-Glycine gel, and (C) SC-1433 depicting a strong DAT signal run on Novex 10% Tris-Glycine gel using mouse striatum sample.

Western blots depicting anti-DAT antibodies with 2^nd extracellular loop immunogens. All western blots were performed using mouse striatum samples from DAT-knock-outs (DKO), and wild-type (WT) mice. Molecular ladder is indicated beside each figure, with detection of the dopamine transporter (DAT) indicated by a black arrow. Western blots using antibodies 2nd loop immunogens include: (A) anti-DAT antibody SC-32259 depicting no DAT signal and weak nonspecific binding on a Novex 10% Tris-Glycine gel using mouse striatum sample, (B) anti-DAT antibody SC-58517 depicting no DAT signal on a Novex 10% Tris-Glycine gel using mouse striatum sample, and (C) anti-DAT antibody 434-DATEL2 depicting no DAT signal and some strong nonspecific binding on a Novex 10% Tris-Glycine gel using mouse striatum sample.

Immunohistology (IH) using antibodies with anti-DAT antibodies that have N-terminal immunogens. IH was performed using wild-type (WT) mouse tissue, DAT-knock-out (DKO) mouse tissue, or rat tissue in which one hemisphere was lesioned with 6-OHDA to kill dopamine neurons, thus eliminating the possibility of a DAT antibody to produce a signal on the lesioned side (internal negative control). Concentration of DAT antibody is indicated in the figure. A, Anti-DAT antibody AB2231 displaying no signal in either mouse or rat tissue at a concentration of 1:100 or 1:200. B, Anti-DAT antibody MAB369 displaying moderate signal in mouse tissue and no signal in rat tissue at a concentration of 1:100 and 1:200. C, Anti-DAT antibody SC-32258 displaying good signal in mouse tissue and no signal in rat tissue at a concentration of 1:100 and 1:200. D, Anti-DAT antibody ZRB1525 displaying a good signal in mouse tissue at a concentration of 1:100 and 1:200, and a poor signal in rat tissue at a concentration of 1:200. E, Anti-DAT antibody PT-22524-1-AP displaying weak signal in mouse tissue at concentrations of 1:100 and 1:200 and no signal in rat tissue at a concentration of 1:100 and 1:200. F, Anti-DAT antibody D6944 showing good signal in mouse tissue at a concentration of 1:200 and a weak signal in rat tissue. G, Anti-DAT antibody MA5-24796 showing good signal in mouse tissue and weak signal in rat tissue at concentrations of 1:100 and 1:200 H, Anti-TH antibody in rat tissue used to demonstrate good detection of the 6-OHDA-lesioned side. Scale bar = 6 mm

High-magnification immunohistology using antibodies with N-terminal immunogens. Immunohistology was performed using wild-type (WT) mouse tissue and DAT-knock-out (DKO) mouse tissue. All DAT antibody concentrations were performed at 1:200. DAPI signal is indicated in blue. A, Anti-DAT antibody ZRB1525 (red) at magnifications of 10×, 20×, and 40×. Clear DAT signal differentiation is seen between WT and DKO with no nonspecific signal (no red signal). B, anti-DAT antibody D6944 stained (red) at magnifications of 10×, 20×, and 40×. Clear DAT signal differentiation is seen WT with no signal in DKO. C, Anti-DAT antibody MA5-24796 (green). Clear DAT signal differentiation is seen between WT and DKO with some nonspecific signal seen in DKO at 40× magnification. Scale bars are indicated in the bottom-left corner of each figure (white).

Immunohistology (IH) using an antibody with a DAT C-terminal immunogens. IH was performed using wild-type (WT) mouse tissue, DAT-knock-out (DKO) mouse tissue, or rat tissue in which one hemisphere was lesioned with 6-OHDA to kill dopamine neurons, thus eliminating the possibility of a DAT antibody to produce a signal on the lesioned side (internal negative control). Concentration of DAT antibody indicated in the figure. A, Anti-DAT antibody 431-DATC displaying a faint signal in WT mouse tissue at a concentration of 1:100 and 1:200 and no signal in rat tissue at either concentration. B, Anti-TH antibody in rat tissue used to demonstrate good detection of the 6-OHDA-lesioned side. Scale bar = 6 mm