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Key Product Details

Species Reactivity

Mouse, Rat, Human (Negative)

Applications

ELISA, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Paraffin, Immunoprecipitation, Western Blot

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG1 Clone # mAb16

Concentration

1.0 mg/ml

Product Summary for SLC6A3/DAT1 Antibody (mAb16)

Immunogen

Synthetic peptide corresponding to the N-terminus of rat DAT1. [UniProt# P23977]

Epitope

N-terminus

Reactivity Notes

Little to no human reactivity has been observed.

Localization

Membrane; Multi-pass membrane protein.

Clonality

Monoclonal

Host

Mouse

Isotype

IgG1

Scientific Data Images for SLC6A3/DAT1 Antibody (mAb16)

Western Blot: SLC6A3/DAT1 Antibody (mAb16) [NBP2-22164] - Western blot analysis of DAT protein. Band shown at approx. 75 kDa. Image submitted by verified customer review.
Immunocytochemistry/Immunofluorescence: DAT1 Antibody (mAb16) [NBP2-22164] - The DAT1 antibody was tested in PC12 cells at a 1:250 dilution against DyLight 488 (Green). Actin and nuclei were counterstained with Phalloidin-AlexaFluor 568 (Red) and DAPI (Blue), respectively.
Immunohistochemistry-Paraffin: DAT1 Antibody (mAb16) [NBP2-22164] - IHC analysis of DAT1 in mouse brain using DAB with hematoxylin counterstain.

Applications for SLC6A3/DAT1 Antibody (mAb16)

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

1:250

Immunohistochemistry

1:100

Immunohistochemistry-Paraffin

1:100

Immunoprecipitation

reported in scientific literature (PMID 20643191)

Western Blot

1:1000
Application Notes
In WB a band can been seen at ~70-85 kDa.
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Reviewed Applications

Read 3 reviews rated 4.7 using NBP2-22164 in the following applications:

Published Applications

Read 14 publications using NBP2-22164 in the following applications:

Formulation, Preparation, and Storage

Purification

Protein G purified

Formulation

Tris-Glycine and 0.15M NaCl

Preservative

0.05% Sodium Azide

Concentration

1.0 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: SLC6A3/DAT1

DAT1, also known as SLC6A3 and Sodium-dependent dopamine transporter, is a sodium and chloride-dependent dopamine transporter belonging to the solute carrier family 6. Dopamine transporters are distributed widely across the brain in regions with high dopaminergic activity, such as the striatum and substantia nigra. These transporters effectively provide fast acting clearance of dopamine, adrenalin and noradrenalin from the synaptic cleft, thus terminating the neurotransmitter signal. DATs have been found to interact with PRKCABP, TGFB1I1, SLC18A2 and the N-terminus of SYNGR3. DATs are also the target proteins for psychomotor stimulants such as amphetamines and cocaine. Research has implicated DATs as useful markers in detecting depression, schizophrenia, Parkinson's disease and drug abuse (Kish et al., 2001; Zhu et al., 2000; Zhu et al., 1999).

Long Name

Dopamine Transporter

Alternate Names

DAT1, SLC6A3, VNTR

Entrez Gene IDs

13162 (Mouse); 24898 (Rat)

Gene Symbol

SLC6A3

Product Documents for SLC6A3/DAT1 Antibody (mAb16)

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot number in the search box below.

Product Specific Notices for SLC6A3/DAT1 Antibody (mAb16)

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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Protocols

View specific protocols for SLC6A3/DAT1 Antibody (mAb16) (NBP2-22164):

[[URL:https://www.novusbio.com/products/slc6a3-dat1-antibody-mab16_nbp2-22164… Antibody (mAb16)]]
Immunocytochemistry Protocol

Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and add 10% formalin to the dish. Fix at room temperature for 30 minutes.
2. Remove the formalin and add ice cold methanol. Incubate for 5-10 minutes.
3. Remove methanol and add washing solution (i.e. PBS). Be sure to not let the specimen dry out. Wash three times for 10 minutes.
4. To block nonspecific antibody binding incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
5. Add primary antibody at appropriate dilution and incubate at room temperature from 2 hours to overnight at room temperature.
6. Remove primary antibody and replace with washing solution. Wash three times for 10 minutes.
7. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
8. Remove antibody and replace with wash solution, then wash for 10 minutes. Add Hoechst 33258 to wash solution at 1:25,0000 and incubate for 10 minutes. Wash a third time for 10 minutes.
9. Cells can be viewed directly after washing. The plates can also be stored in PBS containing Azide covered in Parafilm (TM). Cells can also be cover-slipped using Fluoromount, with appropriate sealing.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.
[[URL:https://www.novusbio.com/products/slc6a3-dat1-antibody-mab16_nbp2-22164… Antibody (mAb16)]]
Immunohistochemistry-Paraffin Embedded Sections

Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes.

Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in wash buffer for 5 minutes.
3. Block each section with 100-400 ul blocking solution for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4 C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul biotinylated diluted secondary antibody. Incubate 30 minutes at room temperature.
7. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
8. Add 100-400 ul Streptavidin-HRP reagent to each section and incubate for 30 minutes at room temperature.
9. Wash sections three times in wash buffer for 5 minutes each.
10. Add 100-400 ul DAB substrate to each section and monitor staining closely.
11. As soon as the sections develop, immerse slides in deionized water.
12. Counterstain sections in hematoxylin.
13. Wash sections in deionized water two times for 5 minutes each.
14. Dehydrate sections.
15. Mount coverslips.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.
[[URL:https://www.novusbio.com/products/slc6a3-dat1-antibody-mab16_nbp2-22164… Antibody (mAb16)]]
Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 40 ug of total protein per lane.
2. Transfer proteins to membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain according to standard Ponceau S procedure (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot.
5. Block the membrane using standard blocking buffer for at least 1 hour.
6. Wash the membrane in wash buffer three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate 1 hour at room temperature.
8. Wash the membrane in wash buffer three times for 10 minutes each.
9. Apply the diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturers instructions) and incubate 1 hour at room temperature.
10. Wash the blot in wash buffer three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturers instructions.
Note: Tween-20 can be added to the blocking or antibody dilution buffer at a final concentration of 0.05-0.2%.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.
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