ADORA1 Rabbit Recombinant mAb | Monoclonal Antibodies

ADORA1 Rabbit Recombinant mAb

Catalog No.A5415

For research use only.

Filter:

  • WB
  • IHC

Usage Information

Application WB, IHC,ELISA
Dilution
WB IHC
1:1000 1:50
Reactivity Human
MW (kDa) 37kDa
Source Rabbit
Concentration 1mg/ml
Storage buffer 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.
Storage Store at –20°C.

Protocol

WB

Western Blotting

Sample preparation

1. Aspirate media from cultures and Wash the cells with 1X PBS.
2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice.
3. Sonicate for 10–15 sec to complete cell lysis and shear DNA.
4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice.
5. Centrifuge for 5 min (with Microcentrifuge).
6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins).
NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer.
7. Electrotransfer to nitrocellulose/PVDF membrane.

Membrane Blocking and Antibody Incubations

a. Blocking

1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature.
2. Incubate the membrane in the blocking buffer for 1 hr at room temperature.
3. Wash three times for 5 min each with TBST.

b. Antibodies Incubation

1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C.
2. Wash three times for 5 min each with TBST.
3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature.
4. Wash three times for 5 min each with TBST.
5. Proceed with detection.

Detection of Proteins

1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST.
2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well.
3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

IHC

Immunohistochemistry (Paraffin)

Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

1. Deparaffinize/hydrate sections:

1. Incubate sections in three washes of xylene for 5 min each.
2. Incubate sections in two washes of 100% ethanol for 10 min each.
3. Incubate sections in two washes of 95% ethanol for 10 min each.

2. Wash sections two times in dH2O for 5 min each.

Antigen retrieval

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.
2. Incubate sections in 3% hydrogen peroxide for 10 min.
3. Wash sections in dH2O two times for 5 min each.
4. Wash sections in wash buffer for 5 min.
5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.
6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.
7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.
9. Wash sections three times with wash buffer for 5 min each.
10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.
11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
12. Immerse slides in dH2O.
13. If desired, counterstain sections with hematoxylin.
14. Wash sections in dH2O two times for 5 min each.
15. Dehydrate sections:
1. Incubate sections in 95% ethanol two times for 10 sec each.
2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
3. Repeat in xylene, incubating sections two times for 10 sec each.
16. Mount sections with coverslips and mounting medium.

Description

Specificity ADORA1 Rabbit Recombinant mAb detects endogenous level of total ADORA1.
Background The protective response to neuron damage is mainly due to endogenous adenosine. Adenosine P1 receptors are a class of purinergic and G protein-coupled receptors using adenosine as a ligand and consist of A1, A2a, A2b, and A3 subtypes. All adenosine receptors have been shown to activate at least one subfamily of mitogen-activated protein kinases. ADORA1 activation inhibits the release of presynaptic transmitters and stabilizes the postsynaptic membrane potential to reduce neuronal transmission and excitability. ADORA2A, another high affinity receptor, is thought to be preferentially activated by a high frequency stimulus, and then strengthen selected synaptic transmission within a globally inhibited network that could be formed by ADORA1 activation. ADORA1 is also an E2/ERα target and a regulator of ERα transcriptional activity.

Datasheet & SDS

Tech Support

If you have any other enquiries, please leave a message.

* Indicates a Required Field

Please enter your name.
Please enter your email. Please enter a valid email address.
Please write something to us.