Anti-Natriuretic Peptide Receptor A / GC-A antibody (ab14356)

Overview

  • Product name
    Anti-Natriuretic Peptide Receptor A / GC-A antibody
    See all Natriuretic Peptide Receptor A / GC-A primary antibodies
  • Description
    Rabbit polyclonal to Natriuretic Peptide Receptor A / GC-A
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IHC-P, ICC, ICC/IF, Flow Cyt, IHC-Frmore details
  • Species reactivity
    Reacts with: Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human Natriuretic Peptide Receptor A/ GC-A aa 294-308 conjugated to keyhole limpet haemocyanin.
    Sequence:

    LKQLKHLAYEQFNFT


    (Peptide available as ab28437, ab49403)

  • General notes

    Previously labelled as Natriuretic Peptide Receptor A. 

Properties

Applications

Our Abpromise guarantee covers the use of ab14356 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 1/5000. Predicted molecular weight: 118 kDa.Can be blocked with Human Natriuretic Peptide Receptor A / GC-A peptide (ab28437).
IHC-P 1/4000.
ICC 1/200.
ICC/IF 1/300.
Flow Cyt 1/200.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

 

IHC-Fr 1/300.

Target

  • Function
    Receptor for the atrial natriuretic peptide NPPA/ANP and the brain natriuretic peptide NPPB/BNP which are potent vasoactive hormones playing a key role in cardiovascular homeostasis. Has guanylate cyclase activity upon binding of the ligand.
  • Sequence similarities
    Belongs to the adenylyl cyclase class-4/guanylyl cyclase family.
    Contains 1 guanylate cyclase domain.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications
    Phosphorylation of the protein kinase-like domain is required for full activation by ANP.
  • Cellular localization
    Membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • ANP-A antibody
    • ANPa antibody
    • ANPR-A antibody
    • ANPRA antibody
    • ANPRA_HUMAN antibody
    • Atrial natriuretic peptide A type receptor antibody
    • Atrial natriuretic peptide receptor 1 antibody
    • Atrial natriuretic peptide receptor A antibody
    • Atrial natriuretic peptide receptor type A antibody
    • Atrionatriuretic peptide receptor A antibody
    • GC A antibody
    • GC-A antibody
    • Guanylate cyclase A antibody
    • Guanylate cyclase antibody
    • GUC2A antibody
    • GUCY2A antibody
    • Natriuretic peptide A type receptor antibody
    • Natriuretic Peptide Receptor A antibody
    • Natriuretic peptide receptor A/guanylate cyclase A antibody
    • NPR-A antibody
    • NPR1 antibody
    • NPRA antibody
    see all

Images

  • ICC/IF image of ab14356 stained MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab14356, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:
  • You H & Laychock SG Long-term treatment with atrial natriuretic peptide inhibits ATP production and insulin secretion in rat pancreatic islets. Am J Physiol Endocrinol Metab 300:E435-44 (2011). WB ; Rat . Read more (PubMed: 20959527) »
  • Matera MG  et al. Epithelium integrity is crucial for the relaxant activity of brain natriuretic peptide in human isolated bronchi. Br J Pharmacol 163:1740-54 (2011). Read more (PubMed: 21410689) »
See all 8 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Application
Flow Cytometry
Sample
Human Cell (HEK293)
Permeabilization
No
Gating Strategy
viability and PE
Specification
HEK293
Preparation
Cell harvesting/tissue preparation method: FACS buffer
Sample buffer: PBS with 1% FBS
Fixation
None

Abcam user community

Verified customer

Submitted Nov 23 2015

Answer

Thank you for your enquiry which we have received through Labome.

For more information regarding ab14356 Natriuretic Peptide Receptor A antibody, I can recommend to review the online datasheet. This will contain all the information we have regarding the product:

https://www.abcam.com/natriuretic-peptide-receptor-a-antibody-ab14356.html

If you have any further specific questions about the peptide, please do not hesitate to contact me again. I will be happy to help.

Regarding information about Abcam, I can recommend to review our website for more information about the products that we sell, and there is also lots of extra information on our various resource pages:
https://www.abcam.com/

We sell primary and secondary antibodies, peptides and proteins, some IHC and WB reagents and kits, and also many detection and activity measurement kits, ChIP kits, ELISPOT kits, and many more. The following page from our website will let you know more about our company:

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

Again, if you have any more specific questions about Abcam, please do not hesitate to let me know.

I hope this will be helpful to you.

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Question
Answer

I received your voicemail and will give you a call. The authors of the recent Neuroscience paper have not replied to me. I examined the original reference: Hirsch JR et al. Cellular localization, membrane distribution, and possible function of guanylyl cyclases A and 1 in collecting ducts of rat. Cardiovasc Res 51:553-61 (2001). PubMed: 11476745 In this case the blot was blocked with Rotiblock for one hour at room temperature, diluted 1:1000 in Rotiblock and incubated overight and the secondary was used at 1:100,000. Yes, 1:100,000. I am not sure what Rotiblock is, as it is not a reagent I am familiar with. I will call you to see what new results you have and then we can decide on a course of action. Also, please email if you would like.

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Answer

I'm sorry to hear you are having a problem with ab14356. Typically, an entirely black blot would mean that there is not enough blocking or far too much protein or antibody. A recent reviewer suggested that the antibody worked well although they did see non-specific bands. You can access this review online on the datasheet. I would like to suggest the following modifications to your protocol: 1) Please load far less protein. We usually load 20-30 ug of protein per lane. I believe 50 to 100 is too much protein. 2) Blocking appears to be fine, although I would recommend blocking in 5% milk for 30 minutes at room temp and also diluting the primary and secondary in 5% milk. 3) I would recommend 4 washing steps after primary and secondary - 1 step for 15 minutes and then 3 washes at 5 min. Also, I assume the TTBS you are using contains Tween, but I wanted to double-check. 4) The secondary antibody can also be diluted further. We would use 1/10,000 to start although in your case you may also want to try more dilute secondary. Please let me know if this helps and do not hesitate to contact us for further advice.

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Question
Answer

Thank you for your enquiry. I have found two papers that use membrane loading controls: 1. BiP, found on ER: www.jbc.org/cgi/content/full/278/36/34725 We have several antiBiP antibodies; I suggest ab2902 https://www.abcam.com/index.html?datasheet=2902 2. Cadherin, a plasma membrane marker: www.nature.com/emboj/journal/v22/n20/full/7590950a.html We have a pan-cadherin antibody I recommend for WB, ab16505 https://www.abcam.com/index.html?datasheet=16505 I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (human aortic endothelial cells)
Loading amount
20 µg
Specification
human aortic endothelial cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%

Brian Van Ness

Verified customer

Submitted Jul 17 2006

Answer

Thank you for your enquiry. I am pleased to report that I have clarified this issue. The immunogen that was used to raise Rabbit polyclonal to Natriuretic Peptide Receptor A (ab14356) was Natriuretic Peptide Receptor A peptide (294-308) (ab28437). I have clarified the immunogen sequence which was amino acids 294-308: LKQLKHLAYEQFNFT. In terms of a blot image I have been informed that the antibody was used in the following publication: Hirsch JR et al. Cellular localization, membrane distribution, and possible function of guanylyl cyclases A and 1 in collecting ducts of rat. Cardiovasc Res 51:553-61 (2001). PubMed: 11476745 You may wish to consult this publication for further information. I have updated our datasheet to reflect this clarification. Good luck with your publication.

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Question

BATCH NUMBER 84581 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal or weak signal and some faint laddering throughout the lane. SAMPLE HeLa cells (human) lysed in M-PER reagent. PRIMARY ANTIBODY Abcam's ab14356 for NPR1, Santa Cruz Biotech's human beta tubulin (mouse) (stock=200ug/ml) and Qiagen's Tag-100 (mouse) (stock=0.2ug/ul) which detects human MAPK1. NPR1 was run at 1:5000 which didn't work. It was re-run at 1:500, 1:1000, 1:2500 and 1:5000 none of which worked. Tubulin was run at 1:100 and Tag-100 at 1:2000 both of which have worked every time they have been used. They were incubated overnight at 4 C in TBS-T + milk. The next day they were washed 4x in TBS or TBS-T. SECONDARY ANTIBODY After the washes, the blot was incubated in TBS-T + milk for 30 minutes with Vectastain's biotinylated universal antibody (Vector Labs) which detects mouse and rabbit. I used the recommended 8 drops in 20mls. After this step the blot was washed 4x in TBS or TBS-T. DETECTION METHOD I used the Vectastain ABC Elite Reagents A+B followed by DAB from Sigma (D-4418) at the recommended concentrations. POSITIVE AND NEGATIVE CONTROLS USED I used a cell lysate from A431 cells (human) provided by the manufacturer of 1 of the antibodies as well as my HeLa cell experimental lysates. ANTIBODY STORAGE CONDITIONS Upon receipt, I aliquoted it and stored it at -20. Each aliquot was thawed and used only once. SAMPLE PREPARATION Samples are in M-PER with protease inhibitors. Samples are reduced and heated at 95 C for 5 minutes and iced. AMOUNT OF PROTEIN LOADED 10 ug and then 20ug ELECTROPHORESIS/GEL CONDITIONS Samples are reduced and run on a 4-12% gel. TRANSFER AND BLOCKING CONDITIONS The gel is transfered to 0.2 PVDF membrane for 1 hr at 30V, then blocked in TBS + 0.1% tween + 5% milk at 4 C for about 3 hours. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? I increased the concentration of the primary NPR antibody from 1:5000 to as high as 1:500 and I increased the amount of protein loaded from 10 to 20ug. ADDITIONAL NOTES Both tubulin and MAPK1 antibodies worked well as they have in the past. I can detect NPR1 mRNA by Taqman analysis and can see knock-down of the RNA after transfecting the cells with an siRNA specific to NPR1 from Dharmacon. It may be that my secondary antibody cannot detect rabbit, though it is supposed to or that my sample preparation conditions are wrong or that the levels of NPR1 protein in HeLa is very low. If you have a lysate of some sort that you have shown to work with this batch of antibody that I could get, that would answer some of these questions.

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Answer

Thank you for your patience. The originator of ab14356 has told me that the antibody was tested with the corresponding antigen for which it was made for. I'm afraid that I'm unable to obtain any more information at this time. Rat kidney membrane samples may be a thought for a positive control ( Hirsch JR et al. Cellular localization, membrane distribution, and possible function of guanylyl cyclases A and 1 in collecting ducts of rat. Cardiovasc Res 51:553-61 (2001). PubMed: 11476745) - this is not the same antibody as ab14356, however. I can offer you a replacement vial of ab14356 to try or I can offer you a refund if you wish. Please let me know which you would prefer.

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Question

BATCH NUMBER 84581 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal or weak signal and some faint laddering throughout the lane. SAMPLE HeLa cells (human) lysed in M-PER reagent. PRIMARY ANTIBODY Abcam's ab14356 for NPR1, Santa Cruz Biotech's human beta tubulin (mouse) (stock=200ug/ml) and Qiagen's Tag-100 (mouse) (stock=0.2ug/ul) which detects human MAPK1. NPR1 was run at 1:5000 which didn't work. It was re-run at 1:500, 1:1000, 1:2500 and 1:5000 none of which worked. Tubulin was run at 1:100 and Tag-100 at 1:2000 both of which have worked every time they have been used. They were incubated overnight at 4 C in TBS-T + milk. The next day they were washed 4x in TBS or TBS-T. SECONDARY ANTIBODY After the washes, the blot was incubated in TBS-T + milk for 30 minutes with Vectastain's biotinylated universal antibody (Vector Labs) which detects mouse and rabbit. I used the recommended 8 drops in 20mls. After this step the blot was washed 4x in TBS or TBS-T. DETECTION METHOD I used the Vectastain ABC Elite Reagents A+B followed by DAB from Sigma (D-4418) at the recommended concentrations. POSITIVE AND NEGATIVE CONTROLS USED I used a cell lysate from A431 cells (human) provided by the manufacturer of 1 of the antibodies as well as my HeLa cell experimental lysates. ANTIBODY STORAGE CONDITIONS Upon receipt, I aliquoted it and stored it at -20. Each aliquot was thawed and used only once. SAMPLE PREPARATION Samples are in M-PER with protease inhibitors. Samples are reduced and heated at 95 C for 5 minutes and iced. AMOUNT OF PROTEIN LOADED 10 ug and then 20ug ELECTROPHORESIS/GEL CONDITIONS Samples are reduced and run on a 4-12% gel. TRANSFER AND BLOCKING CONDITIONS The gel is transfered to 0.2 PVDF membrane for 1 hr at 30V, then blocked in TBS + 0.1% tween + 5% milk at 4 C for about 3 hours. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? I increased the concentration of the primary NPR antibody from 1:5000 to as high as 1:500 and I increased the amount of protein loaded from 10 to 20ug. ADDITIONAL NOTES Both tubulin and MAPK1 antibodies worked well as they have in the past. I can detect NPR1 mRNA by Taqman analysis and can see knock-down of the RNA after transfecting the cells with an siRNA specific to NPR1 from Dharmacon. It may be that my secondary antibody cannot detect rabbit, though it is supposed to or that my sample preparation conditions are wrong or that the levels of NPR1 protein in HeLa is very low. If you have a lysate of some sort that you have shown to work with this batch of antibody that I could get, that would answer some of these questions. My phone number is (858)642-7125.

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Answer

Thank you very much for your patience and for the details that you have provided. I'm currently working with the originator of this antibody and am waiting for some more information from them - in particular, what sort of lysate was used to characterize ab14356 in Western blotting. As soon as I hear back from them (I expect to on Monday), I will let you know. Thanks again.

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