Application

Research CategoryNeuroscience

Anti-Dopamine D1A Receptor Antibody, clone SG2-D1a detects level of Dopamine D1A Receptor & has been published & validated for use in IH & WB.

Research Sub CategoryNeurotransmitters & Receptors

Western blot: 1:250-1:500 on unboiled membrane fractions from rat caudate tissue. Reactivity is also seen in perfrontal and temporal cortex. An identified band between 45-66kDa is observed (Luedtke, 1999).

Immunohistochemistry: 4% PFA fixed, vibratome sections, blocked with 3% NGS, 1% BSA with 0.2% triton X-100. Primary antibodies diluted onlyin PBS with excess blocking reagents.

Optimal working dilutions must be determined by the end user.

SUGGESTED WESTERN BLOT PROTOCOL

Solutions

10X Transfer buffer 1 liter

Tris 115.3 gm

Glycine 24.2 gm

[SDS 4 gm (may be omitted)]

For a 1X working solution: 1 liter

mix 700 mL water

200 mL MeOH

100 mL 10X TransferTBST

stock 1 liter

1M Tris pH 7.5 10 mL

5 M NaC 30 mL

10% Tween-20 5 mL

1. Run SDS-PAGE gel as desired.

2. Soak PVDF membrane (ImmobilonP from Millipore) in 100% methanol for 1-2 minutes to wet the membrane.

3. Soak membrane in transfer buffer (see below) until ready to set up blot transfer (>15 min).

4. Assemble gel-transfer sandwich with the blot toward the positive electrode.

5. Transfer protein from gel to membrane at 100 volts for 1-2 hours for small molecular weight proteins or 65 mA for 16 hours (overnight) for complete transfer of higher molecular weight proteins.

6. Stain the transferred bands with Chemicon BLOT-FastStain™ (Catalog Number 2076).

7. Destain with deionized water.

8. Block with 5% non-fat milk (Marvel or Carnation) in TBS or PBS, overnight at 4°C. The non-fat milk should be dissolved freshly, centrifuged 10,000 rpm for 10 min, and filtered through glass filter (Gelman Acrodisc).

9. Incubation with first antibody overnight with gentle agitation at 4°C.

Optimal working dilutions and incubation time will need to be determined by the end user.

10. Wash at least 3 x 5 min. with TBST. From this stage, azide should be omitted.

11. Incubation with the secondary antibody (HRP-conjugated goat anti-mouse antibody, for example Chemicon Catalog Number AP124P, diluted appropriately) 1 h at room temperature.

12. Wash at least 3 x 5 min. with TBST.

13. Perform ECL with commercial kit (femtoLUCENT, Chemicon Catalog Number 2078).

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Immunogen

Recombinant rat D1a Receptor, carboxy terminus.

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Other Notes

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

Physical form

Purified immunoglobulin. Liquid in 0.02 M Phosphate buffer, 0.25 M NaCl, pH 7.6 with 0.1% sodium azide.

Format: Purified

Specificity

Dopamine Receptor D1A, no cross reactivity to D1B (D5) receptor. Immunoreactivity was found to be specific for mouse and rat D1A receptor. Human, pig, dog, rabbit, sheep and bovine samples were non-reactive by western analysis.

Storage and Stability

Maintain at 2-8°C in undiluted aliquots for up to 6 months after date of receipt.