Overview

  • Product name
    Anti-alpha 1 Adrenergic Receptor antibody
    See all alpha 1 Adrenergic Receptor primary antibodies
  • Description
    Rabbit polyclonal to alpha 1 Adrenergic Receptor
  • Host species
    Rabbit
  • Specificity
    By Western blot, this antibody detects a 60 kDa protein representing the A1AR from mouse kidney membrane preparations, mouse liver, HeLa cell lysates, and zebra fish samples. Immunofluorescence staining of A1AR in mouse kidney distal tubule cells results in staining of the plasma membrane.
  • Tested applications
    Suitable for: WB, ELISA, ICC, ICC/IF, IP, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cat, Human, Drosophila melanogaster, Zebrafish
    Predicted to work with: Rabbit, Guinea pig, Hamster, Cow, Dog, Pig, Japanese ricefish
  • Immunogen

    Synthetic peptide corresponding to Human alpha 1 Adrenergic Receptor aa 339-349 (intracellular). Immunizing peptide corresponds to residues in the 3rd intracellular loop of human A1AR. Thiis sequence is 100% conserved in all A1AR subtypes examined.
    Sequence:

    KFSREKKAAKT


    (Peptide available as ab41797)

  • Positive control
    • mouse kidney membrane preparations, mouse liver lysate, HeLa cell lysate

Properties

Applications

Our Abpromise guarantee covers the use of ab3462 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/400. Detects a band of approximately 60 kDa.Can be blocked with alpha 1 Adrenergic Receptor peptide (ab41797).
ELISA Use at an assay dependent concentration.
ICC 1/10 - 1/1000.
ICC/IF 1/1000.
IP Use at an assay dependent concentration.

use 5 ug/mg lysate

IHC-P 1/50 - 1/200.

Target

Images

  • Immunocytochemistry/Immunofluorescence analysis of alpha 1 Adrenergic Receptor (green) showing staining in the cytoplasm of HepG2 cells (right) compared to a negative control (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with ab3462 in 3% BSA-PBS at a dilution of 1:100 overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

  • ab3462 labelling alpha 1 Adrenergic Receptor in the cytoplasm and membrane of Human prostate tissue (right) compared with a negative control (left). To expose target proteins, antigen retrieval method was performed using 10mM sodium citrate (pH 6.0) microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature. Tissue sections were incubated in the primary antibody (1:100 in 3% BSA-PBS) overnight at 4°C. A HRP-conjugated anti-rabbit was used as the secondary antibody, followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

  • Immunocytochemistry/Immunofluorescence analysis of alpha 1 Adrenergic Receptor (green) showing staining in the cytoplasm of NIH-3T3 cells (right) compared to a negative control (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with ab3462 in 3% BSA-PBS at a dilution of 1:100 overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

  • ab3462 labelling alpha 1 Adrenergic Receptor in the cytoplasm and membrane of Mouse brain tissue (right) compared with a negative control (left). To expose target proteins, antigen retrieval method was performed using 10mM sodium citrate (pH 6.0) microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature. Tissue sections were incubated in the primary antibody (1:100 in 3% BSA-PBS) overnight at 4°C. A HRP-conjugated anti-rabbit was used as the secondary antibody, followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

  • Immunocytochemistry/Immunofluorescence analysis of alpha 1 Adrenergic Receptor (green) showing staining in the cytoplasm of PC-3 cells (right) compared to a negative control (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with ab3462 in 3% BSA-PBS at a dilution of 1:100 overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

  • Immunolocalization of alpha 1 Adrenergic Receptor in mouse distal convoluted tubule using ab3462.

References

This product has been referenced in:
  • Sumi-Ichinose C  et al. Sepiapterin reductase gene-disrupted mice suffer from hypertension with fluctuation and bradycardia. Physiol Rep 5:N/A (2017). Read more (PubMed: 28320892) »
  • de França SA  et al. A Low-Protein, High-Carbohydrate Diet Stimulates Thermogenesis in the Brown Adipose Tissue of Rats via ATF-2. Lipids 51:303-10 (2016). Read more (PubMed: 26781764) »
See all 9 Publications for this product

Customer reviews and Q&As

1-10 of 19 Abreviews or Q&A

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Mouse Tissue sections (heart)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate Buffer
Permeabilization
Yes - 0.2% Triton X-100
Specification
heart
Blocking step
Serum as blocking agent for 10 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Nov 01 2016

Application
Western blot
Sample
Mouse Tissue lysate - other (cellular fractionation of hindpaw skin protein)
Gel Running Conditions
Reduced Denaturing
Loading amount
50 µg
Specification
cellular fractionation of hindpaw skin protein
Blocking step
LiCOR blocking buffer as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 50% · Temperature: 24°C

Abcam user community

Verified customer

Submitted Sep 30 2015

Question
Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Read More

Question
Answer

Thank you for contacting Abcam.

For the antibody ab3462, I was able to locate WB conditions that were used for testing. Please see below.

The samples tested were HeLa cell lysate. rat liver, mouse liver, drosophila and zebra fish. Though I cannot forward the exact image, when 50ug protein loaded on gel and transferred for WB, a strong 60 kDa band was seen for Hela, mouse and zebra fish, a moderate band for Drosophila and a weak band for rat liver.

A ˜24 kDa band was seen for longer film exposure times for HeLa and mouse liver, but this band intensity is very faint and can only be seen when the 60 kDa band is overexposed.

According to the Uniprot website (http://www.uniprot.org/uniprot/P35348), there are multiple isoforms of this protein (in human at least), one of which will be at 38kDa. As to why you detect that band, but lab testing does not, i cannot say. I think the best way to move forward on this, is to replace the current vial of the antibody that you have with ab54730:

https://www.abcam.com/alpha-1-Adrenergic-Receptor-antibody-ab54730.html



Although, we have not tested in in rat tissue, there is a 100% level of homology between the immunogen sequence and rat, therefore I am very confident that it will work.

If this is OK with you, please let me know and I will be have that replacement sent to you. If you could also include the Abcam order number for ab3462 as well, that would be very helpful.

I look forward to your reply.

WB Conditions.
Blocking: 5% Milk, TBS in PBST or TBST (however 5% BSA also tested with good results)
TBST: 0.50M Tris, pH 8.0, 138mM NaCl, 27mM KCl, 0.1% Tween-20
PBST: 10mM Na2HPO4, pH7.2, 138 mM NaCl, 27mM KCL, 0.01%Tween 20

Membrane blocked for 1 hour at room temp.in 20ml Blocking buffer.
Incubate 1:400 dilution primary antibody (diluted in blocking buffer) for 1 hour, RT
Incubate secondary antibody 1:30,000, incubate 1 hour, RT.
Wash membrane 3 x 8 minutes at RT with blocking buffer.
Incubate with ECL substrate. Film time exposures 12 seconds and 30 seconds

Read More

Answer

I am sorry that you have experienced this error! I would be happy to issue a free of charge replacement of the vial of ab3462 you received that did not contain the full volume. This is a mistake on our part, and I certainly want to ensure that you received the full volume that you purchased.

I have just set up an order with the order number of #######. This is currently in stock, and you will receive this on Monday, March 26th.

Please do not hesitate to contact us if you need any additional assistance.

Have a great weekend!

Read More

Answer

Thank you for your inquiry.

I do not suggest to dilute the antibody in the same buffer it is stored in.
The buffer to create the working solutions should be according to the application the antibody is used.

I cannot recommend to add sodium azide to the working solutions in general. Sodium azide inhibits enzyme such as the HRP in WB.

Therefore I suggest to dilute the antibody in PBS only for ICC and in PBS/Tween/ 1% BSA for WB.

This might have to be optimized. For example, if background is seem in WB you might want to increase the amount of BSA to 5%.

General protocols can be found on our extensive protocol pages. Please follow this link:

https://www.abcam.com/index.html?pageconfig=popular_protocols

More information about the storage of antibodies can be found here:

https://www.abcam.com/index.html?pageconfig=resource&rid=10795

I hope this information proves helpful. Please do not hesitate to contact me again with any further questions.

Read More

Answer

Thank you for contacting us.


We believe in providing our customers with all the available information about a product whether that information is positive or negative. However, we do stand behind the quality of this antibody and guarantee that it will work and be specific to the targetin ICC in Mouse, Rat, Human andZebrafish. Ab3462 iscreated using theimmunizing peptide[K(339)FSREKKAAKT(349) ] that corresponds to amino acid residues 339-349 in the 3rd intracellular loop of human A1AR. A Blast of the peptide brings up only alpha 1 Adrenergic Receptors.


Sometimes non-specific binding of an antibody to proteins other than the antigen can sometimes occur. This is usually more common with polyclonal antibodies, but can also occur with monoclonals as well. To determine which band or staining is specificwe offer theimmunizing peptide for blocking experiments. The product code is ab41797 and can be found at the following link:

https://www.abcam.com/alpha-1-adrenergic-receptor-peptide-ab41797.html


I have also attached a document with more information about blocking with an immunizing peptide and a link to our Abpromise guarantee which states that we will Support, Replace or Refund any product should that not perform as stated in the datasheet.


Our Abpromise:
https://www.abcam.com/index.html?pageconfig=resource&rid=10372


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Answer

Thank you for contacting us. I have received more detailed information form the lab: The western blot procedure is as following: Run a SDS-PAGE gel and transfer to membrane. Block in 5% non-fat dry milk in TBS buffer for 1h at RT. Wash with TBST (0.5% Tween-20), incubate in the primary antibody (ab3462, 1:400) for 1h at RT, wash 3x10’ in TBS and then incubate with secondary antibody and detect with substrate. The samples we have tested positive for this antibody are HeLa, mouse liver and Zebra fish (50ug each). The expected band size is slightly smaller than the expected size of ~60kD and we can see a non-specific band appeared at ~97kD. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Answer

Yes, you are right, I double checked the sequence and there is no alpha 1 adrenergic receptor in Drosophila. This was based on WB results as similar size protein was obtained. We have removed Dm and apologize for the confusion. I did a Blast search and the highest homologies were: 1. Dopamine receptor 2 [Drosophila melanogaster]. Predicted size is 60kDa Score = 30.3 bits (64), Expect = 0.022 Identities = 9/11 (82%), Positives = 9/11 (82%), Gaps = 0/11 (0%) Query 1 KFSREKKAAKT 11 KF EKKAAKT Sbjct 410 KFAKEKKAAKT 420 2. 5-hydroxytryptamine receptor 2A [Drosophila melanogaster]. ; Predicted size is 90kDa Score = 24.0 bits (49), Expect = 1.8 Identities = 7/8 (88%), Positives = 7/8 (88%), Gaps = 0/8 (0%) Query 4 REKKAAKT 11 RE KAAKT Sbjct 745 RERKAAKT 752 etc It could be that Dopamine receptor 2 was seen in WB instead. Thank you for pointing this out to us.

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1-10 of 19 Abreviews or Q&A

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