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Adenosine A2aR Antibody (7F6-G5-A2) - BSA Free

Catalog # NBP1-39474 | Novus Biologicals a Bio-Techne Brand
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NBP1-39474
NBP1-39474SS

Key Product Details

Species Reactivity

Human, Mouse, Rat, Porcine, Canine, Guinea Pig, Rabbit, Sheep

Applications

CyTOF-ready, Flow (Intracellular), Flow Cytometry, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin, Western Blot

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG2A Clone # 7F6-G5-A2

Format

BSA Free

Concentration

1.0 mg/ml

Product Summary for Adenosine A2aR Antibody (7F6-G5-A2) - BSA Free

Immunogen

Full length recombinant human Adenosine A2a R. [UniProt# P29274]

Epitope

Epitope mapped to SQPLPGER in the third intracellular loop (PMID 9822147).

Reactivity Notes

Sheep reactivity reported in scientific literature (PMID: 29791204). Please note that this antibody is reactive to Mouse and derived from the same host, Mouse. Mouse-On-Mouse blocking reagent may be needed for IHC and ICC experiments to reduce high background signal. You can find these reagents under catalog numbers PK-2200-NB and MP-2400-NB. Please contact Technical Support if you have any questions.

Clonality

Monoclonal

Host

Mouse

Isotype

IgG2A

Theoretical MW

43 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for Adenosine A2aR Antibody (7F6-G5-A2) - BSA Free

Western Blot: Adenosine A2aR Antibody (7F6-G5-A2) [NBP1-39474] - Total protein from mouse brain was separated on a 12% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 1.0 ug/mL anti-Adenosine A2a R (7F6-G5-A2) in 1% milk, and detected with an anti-mouse HRP secondary antibody using chemiluminescence.
Immunocytochemistry/Immunofluorescence: Adenosine A2aR Antibody (7F6-G5-A2) [NBP1-39474] - HeLa cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton X-100. The cells were incubated with anti-Adenosine A2a R (7F6-G5-A2), NBP1-39474, at a 1:50 dilution overnight at 4C and detected with an anti-mouse Dylight 488 (Green) at a 1:500 dilution. Actin was detected with Phalloidin 568 (Red) at a 1:200 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.
Immunohistochemistry-Paraffin: Adenosine A2aR Antibody (7F6-G5-A2) [NBP1-39474] - Staining of human brain (putamen), antibody at 5 ug/mL.

Applications for Adenosine A2aR Antibody (7F6-G5-A2) - BSA Free

Application
Recommended Usage

Flow (Intracellular)

1 - 5 ug/ml

Flow Cytometry

1-5 ug/mL

Immunocytochemistry/ Immunofluorescence

1:50-1:100. Use reported in scientific literature (PMID 25836920)

Immunohistochemistry

1 -5 ug/ml

Immunohistochemistry-Frozen

reported in scientific literature (PMID 9822147)

Immunohistochemistry-Paraffin

1 -5 ug/ml

Western Blot

1:1000
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Published Applications

Read 17 publications using NBP1-39474 in the following applications:

Formulation, Preparation, and Storage

Purification

Protein G purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% 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: Adenosine A2aR

This gene encodes a protein which is one of several receptor subtypes for adenosine. The activity of the encoded protein, a G-protein coupled receptor family member, is mediated by G proteins which activate adenylyl cyclase. The encoded protein is abundant in basal ganglia, vasculature and platelets and it is a major target of caffeine.

Long Name

Adenosine A2A Receptor

Alternate Names

A2aR, Adenosine A2a R, ADORA2A, RDC8

Entrez Gene IDs

135 (Human); 11540 (Mouse); 25369 (Rat)

Gene Symbol

ADORA2A

Product Documents for Adenosine A2aR Antibody (7F6-G5-A2) - BSA Free

Certificate of Analysis

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

Product Specific Notices for Adenosine A2aR Antibody (7F6-G5-A2) - BSA Free

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 Adenosine A2aR Antibody (7F6-G5-A2) - BSA Free (NBP1-39474):

Protocol for Flow Cytometry Intracellular Staining
Sample Preparation.
1. Grow cells to 60-85% confluency. Flow cytometry requires between 2 x 105 and 1 x 106 cells for optimal performance.
2. If cells are adherent, harvest gently by washing once with staining buffer and then scraping. Avoid using trypsin as this can disrupt certain epitopes of interest. If enzymatic harvest is required, use Accutase, Collagenase, or TrypLE Express for a less damaging option.
3. Reserve 100 uL for counting, then transfer cell volume into a 50 mL conical tube and centrifuge for 8 minutes at 400 RCF.
a. Count cells using a hemocytometer and a 1:1 trypan blue exclusion stain to determine cell viability before starting the flow protocol. If cells appear blue, do not proceed.
4. Re-suspend cells to a concentration of 1 x 106 cells/mL in staining buffer (NBP2-26247).
5. Aliquot out 100 uL samples in accordance with your experimental samples.

Tip: When cell surface and intracellular staining are required in the same sample, it is advisable that the cell surface staining be performed first since the fixation and permeablization steps might reduce the availability of surface antigens.

Intracellular Staining.
Tip: When performing intracellular staining, it is important to use appropriate fixation and permeabilization reagents based upon the target and its subcellular location. Generally, our Intracellular Flow Assay Kit (NBP2-29450) is a good place to start as it contains an optimized combination of reagents for intracellular staining as well as an inhibitor of intracellular protein transport (necessary if staining secreted proteins). Certain targets may require more gentle or transient permeabilization protocols such as the commonly employed methanol or saponin-based methods.
Protocol for Cytoplasmic Targets:
1. Fix the cells by adding 100 uL fixation solution (such as 4% PFA) to each sample for 10-15 minutes.
2. Permeabilize cells by adding 100 uL of a permeabization buffer to every 1 x 106 cells present in the sample. Mix well and incubate at room temperature for 15 minutes.
a. For cytoplasmic targets, use a gentle permeabilization solution such as 1X PBS + 0.5% Saponin or 1X PBS + 0.5% Tween-20.
b. To maintain the permeabilized state throughout your experiment, use staining buffer + 0.1% of the permeabilization reagent (i.e. 0.1% Tween-20 or 0.1% Saponin).
3. Following the 15 minute incubation, add 2 mL of the staining buffer + 0.1% permeabilizer to each sample.
4. Centrifuge for 1 minute at 400 RCF.
5. Discard supernatant and re-suspend in 100 uL of staining buffer + 0.1% permeabilizer.
6. Add appropriate amount of each antibody (eg. 1 test or 1 ug per sample, as experimentally determined).
7. Mix well and incubate at room temperature for 30 minutes- 1 hour. Gently mix samples every 10-15 minutes.
8. Following the primary/conjugate incubation, add 1-2 mL/sample of staining buffer +0.1% permeabilizer and centrifuge for 1 minute at 400 RCF.
9. Wash twice by re-suspending cells in staining buffer (2 mL for tubes or 200 uL for wells) and centrifuging at 400 RCF for 5 minutes. Discard supernatant.
10. Add appropriate amount of secondary antibody (as experimentally determined) to each sample.
11. Incubate at room temperature in dark for 20 minutes.
12. Add 1-2 mL of staining buffer and centrifuge at 400 RCF for 1 minute and discard supernatant.
13. Wash twice by re-suspending cells in staining buffer (2 mL for tubes or 200 uL for wells) and centrifuging at 400 RCF for 5 minutes. Discard supernatant.
14. Resuspend in an appropriate volume of staining buffer (usually 500 uL per sample) and proceed with analysis on your flow cytometer.

Immunocytochemistry Protocol

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

1. Remove culture medium and wash the cells briefly in PBS. Add 10% formalin to the dish and fix at room temperature for 10 minutes.
2. Remove the formalin and wash the cells in PBS.
3. Permeablize the cells with 0.1% Triton X100 or other suitable detergent for 10 min.
4. Remove the permeablization buffer and wash three times for 10 minutes each in PBS. Be sure to not let the specimen dry out.
5. To block nonspecific antibody binding, incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
6. Add primary antibody at appropriate dilution and incubate overnight at 4C.
7. Remove primary antibody and replace with PBS. Wash three times for 10 minutes each.
8. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
9. Remove secondary antibody and replace with PBS. Wash three times for 10 minutes each.
10. Counter stain DNA with DAPi if required.
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 (keep slides in the sodium citrate buffer all the time).

Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in PBS for 5 minutes.
3. Block each section with 100-400 ul blocking solution (1% BSA in PBS) 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 HRP polymer conjugated secondary antibody. Incubate 30 minutes at room temperature.
7. Wash sections three times in wash buffer for 5 minutes each.
8. Add 100-400 ul DAB substrate to each section and monitor staining closely.
9. As soon as the sections develop, immerse slides in deionized water.
10. Counterstain sections in hematoxylin.
11. Wash sections in deionized water two times for 5 minutes each.
12. Dehydrate sections.
13. Mount coverslips.


Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 10-25 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain the membrane with Ponceau S (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot TBS -0.05% Tween 20 (TBST).
5. Block the membrane in 5% Non-fat milk in TBST (blocking buffer) for at least 1 hour.
6. Wash the membrane in TBST three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate overnight at 4C with gentle rocking.
8. Wash the membrane in TBST three times for 10 minutes each.
9. Incubate the membrane in diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) for 1 hour at room temperature.
10. Wash the blot in TBST 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 instructi

FAQs for Adenosine A2aR Antibody (7F6-G5-A2) - BSA Free

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  • Q: What is the  concentration of Adenosine A2a R Antibody (7F6-G5-A2) [Alexa Fluor® 647]

    A: We routinely use 0.1 mg of base antibodies (i.e. unconjugated antibody like NBP1-39474) in our conjugation reactions with the dyes/enzymes of interest. And the recovery rates mostly fall between 85 to 100% and hence the concentrations are about 0.80 - 1.0 mg/ml, in 0.1 ml volume.

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