Recombinant Anti-GABA Transporter 3 / GAT 3 antibody [EPR25153-38] (ab300559)

All lanes : Anti-GABA Transporter 3 / GAT 3 antibody [EPR25153-38] (ab300559) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Rat brain tissue lysate
Lane 3 : Rat thalamus tissue lysate
Lane 4 : Rat spinal cord tissue lysate
Lane 5 : Rat liver tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Performed under non-reducing conditions.

Observed band size: 280,70, 160 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Samples are non-boiled as boiling may cause protein aggregates.

Negative control: Rat liver (PMID: 7854065).

Exposure time: 3 minutes. 

 

70- kDa GAT-3 is observed.  The molecular weight is consistent with what has been described in the literature (PMID: 29742425).

Bands around 160 kDa and 280 kDa may be GAT-3 dimer and tetramer.

 

All lanes : Anti-GABA Transporter 3 / GAT 3 antibody [EPR25153-38] (ab300559) at 1/1000 dilution

Lane 1 : Mouse thalamus tissue lysate at 20 µg
Lane 2 : Mouse liver tissue lysate at 40 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Performed under non-reducing conditions.

Observed band size: 70, 160 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and diluting buffer and concentration: 5% NFDM/TBST. 

This blot was developed using a high sensitivity ECL substrate. 

Samples are non-boiled as boiling may cause protein aggregates.

Negative control: Mouse liver (PMID: 7854065).

70- kDa GAT-3 is observed.  The molecular weight is consistent with what has been described in the literature (PMID: 29742425).

Bands around 160 kDa and 280 kDa may be GAT-3 dimer and tetramer.

Exposure time: 3 minutes. 

Immunohistochemical analysis of paraffin-embedded mouse brain tissue labeling GABA Transporter 3 / GAT 3 with ab300559 at 1/2000 dilution (0.254 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection kit). Positive staining on mouse brain is observed. The section was incubated with ab300559 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody, followed by a ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

Immunohistochemical analysis of paraffin-embedded rat brain tissue labeling GABA Transporter 3 / GAT 3 with ab300559 at 1/2000 dilution (0.254 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection kit). Positive staining on rat brain is observed (PMID: 26390912, 7854065). The section was incubated with ab300559 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody, followed by a ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used. 

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling GABA Transporter 3 / GAT 3 with ab300559 at 1/2000 dilution (0.254 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection kit). Negative control: no staining on mouse liver is observed. The section was incubated with ab300559 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND™ RX instrument. Counterstained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse thalamus (fresh) tissue labeling GABA Transporter 3 / GAT 3 with ab300559 at 1/50 dilution (10.14 μg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 μg/ml) (Green). Positive staining on mouse thalamus is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: PBS was used instead of primary antibody, followed by preadsorbed secondary antibody ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) 1/1000 dilution (2 μg/ml). 

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse liver (fresh) tissue labeling GABA Transporter 3 / GAT 3 with ab300559 at 1/50 dilutio (10.14 μg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 μg/ml) (Green). Negative control: no staining on mouse liver is observed (PMID: 22896705). The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^®488) preadsorbed at 1/1000 dilution (2 μg/ml). 

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat thalamus (fresh) tissue labeling GABA Transporter 3 / GAT 3 with ab300559 at 1/50 dilution (10.14 μg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 μg/ml) (Green). Positive staining on rat thalamus is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: PBS was used instead of primary antibody, followed by a preadsorbed secondary antibody ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (2 μg/ml). 

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Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat liver (fresh) tissue labeling GABA Transporter 3 / GAT 3 with ab300559 at 1/50 dilution (10.14 μg/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 μg/ml) (Green). Negative control: no staining on rat liver is observed (PMID: 22896705). The nuclear counterstain was DAPI (Blue).

Secondary antibody control: PBS was used instead of primary antibody, followed by a preadsorbed secondary antibody ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (2 μg/ml). 

GABA Transporter 3 / GAT 3 was immunoprecipitated from 0.35 mg mouse thalamus tissue lysate with ab300559 at 1/30 dilution (2 μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300559 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse thalamus tissue lysate 10 μg

Lane 2: ab300559 IP in mouse thalamus tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300599 in mouse thalamus tissue lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3.25 seconds

70- kDa GAT-3 band is observed. The molecular weight is consistent with what has been described in the literature (PMID: 29742425). Bands around 160 kDa and 280 kDa may be GAT-3 dimer and tetramer.

GABA Transporter 3 / GAT 3 was immunoprecipitated from 0.35 mg rat thalamus tissue lysate with ab300559 at 1/30 dilution (2 μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300559 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Rat thalamus tissue lysate 10 μg

Lane 2: ab300559 IP in Rat thalamus tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300559 in rat thalamus tissue lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3.25 seconds

70- kDa GAT-3 band is observed. The molecular weight is consistent with what has been described in the literature (PMID: 29742425). Bands around 160 kDa and 280 kDa may be GAT-3 dimer and tetramer.

Observed MW (kDa): 70, 160, 280.