Application

This Anti-Dopamine D1B Receptor Antibody, clone SG4-D1B is validated for use in IH, WB for the detection of Dopamine D1B Receptor.

Research CategoryNeuroscience

Research Sub CategoryNeurotransmitters & Receptors

Western blot: 1:250-1:500 on mouse brain lysate (unboiled sample)

Immunohistochemistry

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

MeOH100 mL

10X Transfer

TBSTstock 1 liter1M

Tris pH 7.5 10 mL

5 M NaCl 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 (>15 min) transfer.

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 water, 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 rocking at 4°C in blocking solution. 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 D1b Receptor, carboxy terminus.

Legal Information

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

Linkage

Replaces: AB1791P

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 D1b (D5).

Storage and Stability

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