Overview

  • Product name

    Anti-Nav1.7 antibody [N68/6]
    See all Nav1.7 primary antibodies
  • Description

    Mouse monoclonal [N68/6] to Nav1.7
  • Host species

    Mouse
  • Specificity

    No cross reactivity against other Nav channels.
  • Tested applications

    Suitable for: ICC/IF, IP, IHC-P, IHC-Fr, Flow Cytmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Fusion protein corresponding to Human Nav1.7 aa 1750-1950 (C terminal).

  • Positive control

    • WB: Cell lysates prepared from CHO cell line constitutively expressing Nav1.7. Flow Cyt: SH-SY5Y cells. IHC: Mouse brain and back skin tissues. IHC-P: Rat and Mouse Dorsal root ganglion tissue sections. ICC/IF: Human NSC derived neurons.
  • General notes

    The clone number has been updated from S68-6 to N68/6, both clone numbers name the same antibody clone.

    This monoclonal antibody is manufactured by Abcam. If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

Applications

Our Abpromise guarantee covers the use of ab85015 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
ICC/IF Use a concentration of 1 - 10 µg/ml.
IP Use at an assay dependent concentration.
IHC-P Use a concentration of 1 - 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
IHC-Fr Use a concentration of 0.1 - 1 µg/ml.
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

Target

  • Function

    Mediates the voltage-dependent sodium ion permeability of excitable membranes. Assuming opened or closed conformations in response to the voltage difference across the membrane, the protein forms a sodium-selective channel through which Na(+) ions may pass in accordance with their electrochemical gradient (PubMed:7720699, PubMed:17167479, PubMed:25240195, PubMed:26680203, PubMed:15385606, PubMed:16988069, PubMed:17145499, PubMed:19369487, PubMed:24311784). It is a tetrodotoxin-sensitive Na(+) channel isoform (PubMed:7720699). Plays a role in pain mechanisms, especially in the development of inflammatory pain (PubMed:17167479, PubMed:17145499, PubMed:19369487, PubMed:24311784).
  • Tissue specificity

    Expressed strongly in dorsal root ganglion, with only minor levels elsewhere in the body, smooth muscle cells, MTC cell line and C-cell carcinoma. Isoform 1 is expressed preferentially in the central and peripheral nervous system. Isoform 2 is expressed preferentially in the dorsal root ganglion.
  • Involvement in disease

    Primary erythermalgia
    Indifference to pain, congenital, autosomal recessive
    Paroxysmal extreme pain disorder
    Generalized epilepsy with febrile seizures plus 7
    Febrile seizures, familial, 3B
  • Sequence similarities

    Belongs to the sodium channel (TC 1.A.1.10) family. Nav1.7/SCN9A subfamily.
    Contains 1 IQ domain.
  • Domain

    The sequence contains 4 internal repeats, each with 5 hydrophobic segments (S1,S2,S3,S5,S6) and one positively charged segment (S4). Segments S4 are probably the voltage-sensors and are characterized by a series of positively charged amino acids at every third position.
  • Post-translational
    modifications

    Phosphorylation at Ser-1490 by PKC in a highly conserved cytoplasmic loop increases peak sodium currents.
    Ubiquitinated by NEDD4L; which may promote its endocytosis. Does not seem to be ubiquitinated by NEDD4.
  • Cellular localization

    Cell membrane. Cell projection. In neurite terminals.
  • Information by UniProt
  • Database links

  • Alternative names

    • ETHA antibody
    • GEFSP7 antibody
    • hNE Na antibody
    • hNE-Na antibody
    • hNENa antibody
    • NE NA antibody
    • NENA antibody
    • Neuroendocrine sodium channel antibody
    • Peripheral sodium channel 1 antibody
    • PN1 antibody
    • Scn9a antibody
    • SCN9A_HUMAN antibody
    • Sodium channel protein type 9 subunit alpha antibody
    • Sodium channel protein type IX subunit alpha antibody
    • Sodium channel voltage gated type IX alpha antibody
    • Sodium channel voltage gated type IX alpha polypeptide antibody
    • Sodium channel voltage gated type IX alpha subunit antibody
    • Voltage gated sodium channel alpha subunit Nav1.7 antibody
    • Voltage gated sodium channel subunit alpha Nav1 antibody
    • Voltage-gated sodium channel subunit alpha Nav1.7 antibody
    see all

Images

  • Paraformaldehyde-fixed human NSC derived neurons stained for Nav1.7 (red) using ab85015 at 1/200 dilution in ICC/IF, followed by Donkey Anti-Mouse IgG (H+L) Cy3® at 1/100 dilution.

    See Abreview

  • IHC image of Nav1.7 staining in mouse dorsal root ganglion formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab85015, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • IHC image of Nav1.7 staining in rat dorsal root ganglion formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab85015, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • Overlay histogram showing SH-SY5Y cells stained with ab85015 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab85015, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive result in SH-SY5Y cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween for 20 min used under the same conditions.

  • ab85015 at 1/100 dilution staining Nav1.7 in mouse back skin tissue section by IHC-P. Bouin's fixed and paraffin-embedded tissue sections were used. Tissue underwent heat mediated antigen retrieval in microwave with two, 5 minutes incubation intervals in citrate buffer. A Fluorophore conjugated goat anti mouse at 1/50 dilution was used as secondary.

References

This product has been referenced in:

  • Hofmann L  et al. Characterization of small fiber pathology in a mouse model of Fabry disease. Elife 7:N/A (2018). Read more (PubMed: 30328411) »
  • Bi RY  et al. Estradiol upregulates voltage-gated sodium channel 1.7 in trigeminal ganglion contributing to hyperalgesia of inflamed TMJ. PLoS One 12:e0178589 (2017). IHC-Fr ; Rat . Read more (PubMed: 28582470) »
See all 2 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (NSC derived neurons)
Permeabilization
Yes - Triton X-100
Specification
NSC derived neurons
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Fixative
Paraformaldehyde

Mr. Thomas Klein

Verified customer

Submitted Nov 13 2015

Question
Answer

The specificity of these antibodies to their target Nav channel was determined by performing western blot analysis on lysates from various HEK cell lines expressing one Nav channel. These antibodies were used to detect the presence of the channel in the lysates, and it was determined that ab85015 only detects Nav1.7 and ab93616 only detects Nav1.8.
Both these products were tested in the neuroblastoma cell line SH-SY5Y due to expression in dorsal root ganglion. These products are both tested and guaranteed to be specific for their respective Nav channels in the applications and species listed on their datasheets.

Read More

Answer

Thank you for your response and for taking the time for providing some useful information.

After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:

Transfection:

Have you checked the efficiency of transfection?

Does the construct contain full sequence or fragment of the target protein?

Have you used any tagging which may interact with the recognition of the epitope?

Positive control:

It would be worth running a good positive (cells or tissues which endogenously express Nav1.7) control along with the samples. For this purpose, HEK293 or samples from spinal cord can be used. Nav1.7 is expressed strongly in dorsal root ganglion, with only minor levels elsewhere in the body, smooth muscle cells, MTC cell line and C-cell carcinoma.

Isoforms

Do you know which isoform(s) is expressed in the samples? It has been reported that Isoform 1 is expressed preferentially in the central and peripheral nervous system. Isoform 2 is expressed preferentially in the dorsal root ganglion.

I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

Read More

Answer

Thank you for getting back to me.
although it may be beneficial to optimise the transfer as far as possible (and I would still suggest staining the membrane to observe the protein transfer if possible), you have seen some signal within the expected range so this may not be the most crucial point.
I think it will be important to check that the protein is within the soluble fraction and not discarded following centrifugation. Additionally, as you have been seeing a large number of smaller protein fragments, ensuring the protein is not broken down by adding additional protease inhibitors may be beneficial.
Please do let me know how you get on and if you have any further questions.
Until then, I wish you all the best with your experiments.

Read More

Answer

Thank you for confirming those details.
I have a few suggestions which may help improve the results observed with ab85015.
The target you are hoping to detect is quite tricky as it is large (expected ˜226 kDa) and membrane bound. Some optimisation in order to get a good signal may be required. I would suggest trying the following:
1. I would suggest lysing the cells for a little longer to ensure complete lysis (up to 30 minutes with agitation). If you have access to a sonicator this may also improve the lysis. Make sure to keep the samples on ice throughout to reduce the level of proteolysis.
2. I would suggest adding further protease inhibitors to your sample. The blot presented suggests that the protein is being degraded and several smaller bands is being detected. Using PMSF will only reduce serine and cystein protease activity. In order to reduce degradation I would add at least add 5 mM EDTA and pepstatin A if you have access to them. Ideally a protease cocktail, widely available, such as ab65621 should be used.
3. I would suggest increasing the amount of protein loaded from 10 ug to at least 30 ug. Additionally, in order to ascertain if the protein has remained membrane bound following lysis, I would suggest also loading a sample of the cell debris collected after centrifugation.
4. When I enquired after if the protein transfer had been ascertained I meant if the membrane had been stained using something like ponceau red. This would be especially important with this protein as it is large and transfer conditions will need to be optimised. It will also indicate if the protein is being degraded as the transfected and over expressed protein should be detectable. Transfer of larger proteins can be improved by including SDS to a final concentration of 0.1% in the transfer buffer. Reducing the amount of methanol in the buffer can also help. 10% can be used, or if using PVDF methanol can be removed from the buffer altogether. This aids swelling of the gel, allowing proteins to transfer more easily. The PVDF membrane does still need to be activated in methanol however.
5. I would suggest running a "no primary" control to ascertain if the non-specific bands observed are contributed to from the secondary antibody. The use of a loading control in this instance may also be useful as this will give an indication of the quality of the sample.
I hope this information has been of help. If you require any further information, please do not hesitate to ask.

Read More

Answer

Thank you for contacting us and updating us on the progress you have been making.
xxxxx is unfortunately away from the office at present but I will try to help as best I can on her behalf.
I'm please do hear that the antibody has been working well in flow cytometry. It is unfortunate that the Western blot results have not been as good. In order to understand the problems encountered a little further , would it be possible for you to share an image of the blot obtained?
I also have some additional questions:
1. How much protein was loaded into each well?
2. Was the protein transfer verified by staining of the membrane?
3. Was the secondary antibody used the same as that which was used for the flow cytometry?
4. Was any other protein detected in the samples, i.e. was a different primary antibody used to detect a loading control protein such as tubulin?
With these details I will hopefully understand more fully what may be contributing to the problems encountered.
I have contacted the source of this antibody to obtained a detailed description of how the antibody has previously been used in Western blotting and I will share this with you as soon as I receive it.
I look forward to receiving your reply.

Read More

Answer

Thank you for your response.
We do not have specific protocol for ICC or ICC/IF for intracellular staining of adherent cells (HEK, PC12) in a flat bottom 96-well plate.
We have some general protocols under our Protocol site which could be altered or optimized.
https://www.abcam.com/index.html?pageconfig=resource&rid=11462
https://www.abcam.com/index.html?pageconfig=resource&rid=11417
I hope this helps and if I can assist further, please do not hesitate to contact me.

Read More

Answer

Thank you for your response.
My colleague is out of office this week and she has asked me to look after her customers.
I have attached a protocol used for testing this particular antibody (ab85015). In our Lab, we have not tested it by ourselves and it was characterized successfully for this assay by our collaborators.
I hope this helps and if I can assist further, please do not hesitate to contact me.

Read More

Answer

Thank you for your reply and further questions which have been forwarded to me as my colleague Karin is currently away from the office.

In answer to your questions regarding the protocol:

1. Centrifuging for Cell Fixation is 500 x g. Centrifuging cell staining is 800 x g. The main reason for this increase is that fixed cells may need to be pelleted harder. Our protocol uses 96-well plates and if not pelleted at this speed then we usually end up with fewer to analyse by the end of our staining protocol as some are lost when removing wash buffer.

2. In our lab protocol, permeabilisation is performed at room temperature.

3. Tween-20 provides sufficient permeabilisation – we have validated this protocol for use within our lab and it works well. We recommend to include saponin in all the buffers following permeabilisation, as once saponin is removed the membrane pores will close as it works by interacting with cholesterol in the membrane.

I hope this will be helpful. If you have any further questions, please do not hesitate to contact us.

Read More

Answer

Thank you for getting back to me.
The wash buffer used following isolation of the cells and before fixation was just PBS. Following fixation and permeabilization, 1x PBS/1% Foetal Bovine serum was used to was the cells. I see no reason why this protocol should not work for staining of adherent cells.
If you have any further problems or questions please do not hesitate to contact us again.

Read More

1-10 of 12 Abreviews or Q&A

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