Recombinant Anti-Dopamine Transporter antibody [EPR19695] (ab184451)

All lanes : Anti-Dopamine Transporter antibody [EPR19695] (ab184451) at 1/1000 dilution

Lane 1 : Mouse striatum lysate boiled
Lane 2 : Mouse striatum lysate unboiled
Lane 3 : Rat striatum lysate boiled
Lane 4 : Rat striatum lysate unboiled

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 69 kDa

We recommend not to boil the samples after lysis to get desired WB results

All lanes : Anti-Dopamine Transporter antibody [EPR19695] (ab184451) at 1/1000 dilution

Lane 1 : Mouse striatum lysate
Lane 2 : Rat striatum lysate
Lane 3 : Mouse kidney lysate
Lane 4 : Rat liver lysate
Lane 5 : Neuro-2a (Mouse neuroblastoma cell line) whole cell lysate
Lane 6 : C6 (Rat glial tumor cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 69 kDa
Observed band size: 70-85 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Dopamine transporters are distributed widely across the brain in regions with high dopaminergic activity, such as the striatum and substantia nigra (PMID:1765147), so non-related tissues and cells were chosen as negative controls; and DAT is a glycoprotein with molecular weight of 70-85KD (PMID:25356398, PMID: 20643191).

IHC image of Dopamine Transporter staining in a section of formalin fixed paraffin embedded normal human striatum* performed on a Leica BOND™.  The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab184451, 5ugml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen.

The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

IHC image of Dopamine Transporter staining in a section of formalin fixed paraffin embedded normal human substantia nigra* performed on a Leica BOND™.  The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was incubated with ab184451, 5ugml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Negative control image: IHC image of Dopamine Transporter staining in a section of formalin fixed paraffin embedded normal human liver* performed on a Leica BOND™.  The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins..The section was incubated with ab184451, 5ugml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Dopamine Transporter was immunoprecipitated from 0.35mg of Mouse striatum whole cell lysate with ab184451 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab184451 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: Mouse striatum whole cell lysate, 10µg (Input).

Lane 2: ab184451 IP in Mouse striatum whole cell lysate.

Lane 3: Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab184451 in Mouse striatum whole cell lysate.

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

Exposure time: 30 seconds.