Overview

  • Product name
    Anti-nNOS (neuronal) (phospho S847) antibody
    See all nNOS (neuronal) primary antibodies
  • Description
    Rabbit polyclonal to nNOS (neuronal) (phospho S847)
  • Host species
    Rabbit
  • Specificity
    This antibody shows a reduction in signal when blocked with unmodified nNOS (neuronal) peptide in WB, however when tested in ELISA, it showed less than 2% cross reactivity with the unmodified protein.
  • Tested applications
    Suitable for: IHC-Fr, IHC-FoFr, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Apteronotus leptorhynchus
    Predicted to work with: Rabbit, Human, Xenopus laevis, Zebrafish
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 800 - 900 of Mouse nNOS (neuronal), phosphorylated at S847.

    Read Abcam's proprietary immunogen policy (Peptide available as ab16981.)

  • Positive control
    • This antibody gave a positive signal in mouse forebrain and mouse spinal cord tissue lysates. HeLa whole cell lysate was used as a negative control. nNOS is widely expressed in the nervous system: cerebrum, olfactory bulb, hippocampus, midbrain, cerebellum, pons, medulla oblongata, and spinal cord. It is also found in skeletal muscle, spleen, heart, kidney, and liver.

Properties

Applications

Our Abpromise guarantee covers the use of ab16650 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
IHC-Fr Use at an assay dependent concentration.
IHC-FoFr 1/3000.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 160 kDa (predicted molecular weight: 160 kDa).

Target

  • Function
    Produces nitric oxide (NO) which is a messenger molecule with diverse functions throughout the body. In the brain and peripheral nervous system, NO displays many properties of a neurotransmitter. Probably has nitrosylase activity and mediates cysteine S-nitrosylation of cytoplasmic target proteins such SRR.
  • Tissue specificity
    Isoform 1 is ubiquitously expressed: detected in skeletal muscle and brain, also in testis, lung and kidney, and at low levels in heart, adrenal gland and retina. Not detected in the platelets. Isoform 3 is expressed only in testis. Isoform 4 is detected in testis, skeletal muscle, lung, and kidney, at low levels in the brain, but not in the heart and adrenal gland.
  • Sequence similarities
    Belongs to the NOS family.
    Contains 1 FAD-binding FR-type domain.
    Contains 1 flavodoxin-like domain.
    Contains 1 PDZ (DHR) domain.
  • Domain
    The PDZ domain in the N-terminal part of the neuronal isoform participates in protein-protein interaction, and is responsible for targeting nNos to synaptic membranes in muscles. Mediates interaction with VAC14.
  • Post-translational
    modifications
    Ubiquitinated; mediated by STUB1/CHIP in the presence of Hsp70 and Hsp40 (in vitro).
  • Cellular localization
    Cell membrane > sarcolemma. Cell projection > dendritic spine. In skeletal muscle, it is localized beneath the sarcolemma of fast-twitch muscle fiber by associating with the dystrophin glycoprotein complex. In neurons, enriched in dendritic spines.
  • Information by UniProt
  • Database links
  • Alternative names
    • 2310005C01Rik antibody
    • BNOS antibody
    • Constitutive NOS antibody
    • EC 1.14.13.39 antibody
    • IHPS 1 antibody
    • IHPS1 antibody
    • N-NOS antibody
    • NC-NOS antibody
    • neuronal Nitric Oxide Synthase antibody
    • Neuronal NOS antibody
    • Nitric oxide synthase , neuronal, included antibody
    • Nitric oxide synthase 1 (neuronal) antibody
    • Nitric oxide synthase 1 antibody
    • Nitric oxide synthase, brain antibody
    • Nitric oxide synthase, penile neuronal, included antibody
    • NNOS antibody
    • NO antibody
    • NOS 1 antibody
    • NOS antibody
    • NOS type I antibody
    • NOS-I antibody
    • NOS1 antibody
    • NOS1_HUMAN antibody
    • Peptidyl-cysteine S-nitrosylase NOS1 antibody
    see all

Images

  • All lanes : Anti-nNOS (neuronal) (phospho S847) antibody (ab16650) at 1 µg/ml

    Lane 1 : Forebrain (Mouse) Tissue Lysate
    Lane 2 : Spinal Cord (Mouse) Tissue Lysate
    Lane 3 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (negative control)
    Lane 4 : Forebrain (Mouse) Tissue Lysate with Mouse nNOS (neuronal) (phospho S847) peptide at 1 µg/ml
    Lane 5 : Spinal Cord (Mouse) Tissue Lysate with Mouse nNOS (neuronal) (phospho S847) peptide at 1 µg/ml
    Lane 6 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (negative control) with Mouse nNOS (neuronal) (phospho S847) peptide at 1 µg/ml
    Lane 7 : Forebrain (Mouse) Tissue Lysate with Mouse nNOS (neuronal) (unmodified) peptide at 1 µg/ml
    Lane 8 : Spinal Cord (Mouse) Tissue Lysate with Mouse nNOS (neuronal) (unmodified) peptide at 1 µg/ml
    Lane 9 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (negative control) with Mouse nNOS (neuronal) (unmodified) peptide at 1 µg/ml

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 160 kDa
    Observed band size: 190 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 55 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 4 minutes


    This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab16650 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

  • All lanes : Anti-nNOS (neuronal) (phospho S847) antibody (ab16650) at 1 µg/ml

    Lane 1 : Forebrain (Mouse) Tissue Lysate
    Lane 2 : Spinal Cord (Mouse) Tissue Lysate
    Lane 3 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (negative control)

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 160 kDa
    Observed band size: 190 kDa why is the actual band size different from the predicted?


    Exposure time: 2 minutes
  • Lanes 1-3 : Anti-nNOS (neuronal) (phospho S847) antibody (ab16650) at 1 µg/ml
    Lane 4 : nNOS antibody at 1/2500 dilution

    Lanes 1 & 4 : mouse forebrain
    Lane 2 : mouse forebrain with Mouse nNOS (neuronal) (phospho S847) peptide (ab16981) at 1 µg/ml
    Lane 3 : mouse forebrain with corresponding unmodified nNOS (neuronal) peptide at 1 µg/ml

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 160 kDa
    Observed band size: 160 kDa

  • Immunostaining using Rabbit polyclonal to nNOS (neuronal) (phospho S847) (ab16650) on rat brain tissue sections (30 micron free floating). ab16650 was used at a dilution of 1/3000 and incubated for 18 hours at RT (in PBS triton 0.3%). Secondary Antibody Goat anti-rabbit alexa Fluor 488 was used at a dilution of 1/1000. The image shows cytoplasmic staining of CNS neurons with ab16650 in naïve rats; the staining being observed in the soma and processes of these neurons. The staining was quenched by pre-incubation with peptide against phospho S847 (ab16981), but not by the control peptide (ab57047) indicating that ab16650 is specific for nNos phosphorylated at S847. Protocol: Rats were perfused-fixed with 4% paraformaldehyde. Tissues were post-fixed overnight in the same fixative and then cryoprotected in 20% sucrose overnight. Following embedding in OCT and freezing, tissues were cut and immunostained using the 'free floating’ technique.
  • ab16650 staining nNOS (neuronal) (phospho S847) in 16 µm thick sections of Apteronotus leptorhynchus by Immunohistochemistry (Frozen sections).
    Tissue was fixed in 2% paraformaldehyde, permeabilized using 0.3% Triton X-100, blocked with 3% sheep serum, 1% BSA, 1% teleostean gelatine in TBS for 1 hour at 24°C and then incubated with ab16650 at a 1/100 dilution for 18 hours at 4°C. The secondary used was an Alexa-Fluor 488 conjugated goat anti-rabbit polyclonal used at a 1/200 dilution.

    See Abreview

References

This product has been referenced in:
  • Llévenes P  et al. Thyroid hormones affect nitrergic innervation function in rat mesenteric artery: Role of the PI3K/AKT pathway. Vascul Pharmacol N/A:N/A (2018). WB . Read more (PubMed: 29751093) »
  • Smith ACW  et al. Accumbens nNOS Interneurons Regulate Cocaine Relapse. J Neurosci 37:742-756 (2017). Read more (PubMed: 28123012) »
See all 10 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Apteronotus leptorhynchus Tissue lysate - whole (Brain)
Loading amount
50 µg
Specification
Brain
Gel Running Conditions
Reduced Denaturing (4-15%)
Blocking step
Milk as blocking agent for 14 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Sep 28 2012

Answer

Thank you for the extra details of your protocol. I think there is nothing wrong with your protocol. I was concerned that you may have been using milk to block the membrane and dilute the antibody, since this can sometimes interfere with detection of phospho-proteins. The Odyssey blocking buffer does not, so I think we should consider replacing this antibody.

Can you please send me the order number (either a PO number or the Abcam reference number), or the name of the person who placed the order, the institution, and the approximate date?

Read More

Answer

Thank you for bringing this to our attention.

Can you please confirm that you are using ab16650?

I think there are too many bands to identify which is nNOS, but if you send a few details of your western blot protocol, I may be able to suggest a modification that improves the result.

Can you please tell me:

1. How much sample you load per lane of the gel?
2. What you are using to block the membrane?
3. What you dilute the antibody in?
4. What concentration or dilution of antibody you incubate with the membrane?

I look forward to your reply.

Read More

Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimise the results from ab16650 Anti-nNOS (neuronal) (phospho S847) antibody. I would also appreciate if you can confirm some further details:


1.As you are fixing with PFA and overnight, a gentle antigen retrieval may be necessary. Please reffer to the following http://www.elsevier.com/wps/find/journaldescription.cws_home/506079/description#description. 2000 Dec 15;104(1):27-34.

2. What kind of incubation buffer are you using? Does it contain a detergent like Tween or Triton? If you don't Iwould like to suggest toadd 0.025% Triton X-100 to facilitate the antibody solution.

3. I would like to suggest to block the endogenous biotin as you are working with the ABC system. this may explain the high background of the old lot.

4.Have you alternated the antibodies concentration? According to the datasheet, the antibodies concentration fluctuates form lot to lot.


Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Apteronotus leptorhynchus Tissue sections (Brain)
Specification
Brain
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.3% Triton X-100
Blocking step
3% sheep serum, 1% BSA, 1% teleostean gelatine in TBS as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 24°C

Dr. Ruxandra Sirbulescu

Verified customer

Submitted Nov 18 2011

Question
Answer

I am very pleased to hear you would like to accept our offer and test ab16650 and ab5583 in electric fish. This code will give you 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview for electric fish and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews. Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code. Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research. The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Tissue lysate - other (Mouse cortex)
Loading amount
15 µg
Specification
Mouse cortex
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Oct 05 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Rat Tissue sections (Brain sections (30 um free floating))
Specification
Brain sections (30 um free floating)
Fixative
Paraformaldehyde
Antigen retrieval step
None
Permeabilization
No

Dr. Sophie Pezet

Verified customer

Submitted Jan 17 2008

Answer

Thank you for the protocol details that you have provided. I would expect you to see a band at approximately 160 kDa using mouse forebrain samples and using ab16650 at a concentration of 1 ug/ml. Since you are not seeing anything at all, even with the primary at 2 ug/ml, incubation o/n at 4C and up to 80 ug of lysate loaded, it definitely seems as if there is a problem with the vial that you received. I just want to check, do you know that your secondary is working properly with other primaries? I would be happy to offer you a free of charge replacement vial or can offer to issue you a credit note or refund. Please let me know how you would like to proceed and I look forward to hearing from you.

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