Analysis Note

ControlIncluded free of charge with the antibody is 40 µg of control antigen. The stock solution of the antigen can be made up using 100 µL of sterile distilled water. For negative control, preincubate 1 µg of peptide with 1 µg of antibody for one hour at room temperature. Optimal concentrations must be determined by the end user.

Application

Western blot: 1:2000 using ECL on rat brain lysate.

Dilutions should be made using a carrier protein such as BSA (1-3%)

Optimal working dilutions must be determined by the end user.

SUGGESTED WESTERN BLOT PROTOCOL

1. Mix the samples (organ membranes: 50 µg/lane; transfected cells: 500,000 cells/lane) with sample-buffer X 2, and heat 10 min at 70°C.

2. 5-50 µL applied to Minigel lane (0.75-1.5 mm width) and run at standard conditions. (60 mA for 2 1.5 mm Minigel gels, 1.4 h). It is suggested that you run 5-15% acrylamide (37.5:1 acrylamide:bisacrysmide) minigel (1.5 mm width) at 30 mA/gel ~1-1.5 hours.

3. Transfer in semi-dry system under standard conditions (3 h 100 mA for two minigel gels)

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

5. Destain with deionized water.

6. Block with 5% non-fat milk (Marvel or Carnation) in PBS, and 0.025 % sodium azide, overnight at 2-8°C. The non-fat milk should be dissolved freshly, centrifuged 10,000 rpm for 10 min, and filtered through glass filter (Gelman Acrodisc).

7. Incubation with first antibody 2 h at room temperature or overnight at 4°C in blocking solution. The antibody preparation should be centrifuged before use (10,000 g 5 min.). Optimal working dilutions and incubation time will need to be determined by the end user.

8. Wash 4 x 10 min. with PBS-0.1% tween 20. From this stage, azide should be omitted.

9. Incubation with the secondary antibody (HRP-conjugated goat anti-rabbit antibody, for example Chemicon Catalog Number AP132P, diluted appropriately) 1 h at room temperature.

10. Wash 4 x 10 min. with PBS-0.1% tween 20.

11. Perform ECL with commercial kits (Chemilucent, Chemicon Catalog Number 2600).

Research CategoryNeuroscience

Research Sub CategoryNeurotransmitters & Receptors

This Anti-Muscarinic Acetylcholine Receptor m3 Antibody is validated for use in WB for the detection of Muscarinic Acetylcholine Receptor m3.

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

Synthetic peptide from the 3rd intracellular loop of rat m3 (Accession P08483). The immunogen sequence is identical in human, mouse, bovine and porcine.

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

Affinity purified immunoglobulin. Lyophilized from phosphate buffered saline, pH 7.4, containing 1% BSA, and 0.05% sodium azide as a preservative. Reconstitute with 50 µL of sterile deionized water. Centrifuge antibody preparation before use (10,000 xg for 5 min).

Specificity

Muscarinic acetylcholine receptor m3.

Storage and Stability

Maintain lyophilized material at -20°C for up to 12 months after date of receipt. After reconstitution maintain at -20°C in undiluted aliquots for up to 6 months. Avoid repeated freeze/thaw cycles.