Overview

  • Product name

    Anti-Nav1.6/SCN8A antibody
    See all Nav1.6/SCN8A primary antibodies
  • Description

    Rabbit polyclonal to Nav1.6/SCN8A
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IHC-FrFl, ICC/IF, IHC-P, IHC-Fr, WB, IHC-FoFrmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human Nav1.6/SCN8A.

  • General notes

     This product was previously labelled as Nav1.6

     

Properties

Applications

Our Abpromise guarantee covers the use of ab65166 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-FrFl Use at an assay dependent concentration. See Abreview.
ICC/IF Use at an assay dependent concentration. See Abreview.
IHC-P Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.
WB 1/500 - 1/2000. Predicted molecular weight: 220 kDa.
IHC-FoFr Use at an assay dependent concentration. PubMed: 27905510

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. In macrophages and melanoma cells, isoform 5 may participate in the control of podosome and invadopodia formation.
  • Tissue specificity

    Isoform 5 is expressed in non-neuronal tissues, such as monocytes/macrophages.
  • Sequence similarities

    Belongs to the sodium channel (TC 1.A.1.10) family. Nav1.6/SCN8A 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

    May be ubiquitinated by NEDD4L; which would promote its endocytosis.
  • Cellular localization

    Membrane and Cytoplasmic vesicle. Some vesicles are localized adjacent to melanoma invadopodia and macrophage podosomes. Does not localize to the plasma membrane.
  • Information by UniProt
  • Database links

  • Alternative names

    • CerIII antibody
    • CIAT antibody
    • EIEE13 antibody
    • hNa6/Scn8a voltage gated sodium channel antibody
    • MED antibody
    • Motor endplate disease antibody
    • NaCh 6 antibody
    • NaCh6 antibody
    • Nav 1.6 antibody
    • Nbna1 antibody
    • peripheral nerve protein type 4 antibody
    • PN 4 antibody
    • PN4 antibody
    • SCN8A antibody
    • SCN8A_HUMAN antibody
    • Sodium channel protein type 8 alpha subunit antibody
    • Sodium channel protein type 8 subunit alpha antibody
    • Sodium channel protein type VIII alpha subunit antibody
    • Sodium channel protein type VIII subunit alpha antibody
    • Sodium channel voltage gated type VIII alpha antibody
    • Sodium channel voltage gated type VIII alpha polypeptide antibody
    • Sodium channel voltage gated type VIII alpha subunit antibody
    • Voltage gated sodium channel subunit alpha Nav1.6 antibody
    • Voltage-gated sodium channel subunit alpha Nav1.6 antibody
    see all

Images

  • PFA perfusion-fixed sections of rat hippocampus (following 21 fdays of KA treatment) using ab65166 at 1/600 dilution (red). Co-localization with Nestin (green) is shown.

    Data taken from PMID 27905510 (Figure 7E).

    Remarkable alterations of Nav1.6/SCN8A in reactive astrogliosis during epileptogenesis
    HongyanZhu, Yuxiao Zhao*, HaoWu, Nan Jiang, ZiyWang, Weide Lin, Jiahui Jin & Yonghua Ji

    Scientific Reports | 6:38108 | DOI: 10.1038/srep38108

    This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

References

This product has been referenced in:

  • Arechederra M  et al. Hypermethylation of gene body CpG islands predicts high dosage of functional oncogenes in liver cancer. Nat Commun 9:3164 (2018). Read more (PubMed: 30089774) »
  • Zhu H  et al. Remarkable alterations of Nav1.6 in reactive astrogliosis during epileptogenesis. Sci Rep 6:38108 (2016). IHC-P ; Rat . Read more (PubMed: 27905510) »
See all 5 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (fetal spinal cord nerve fibers)
Antigen retrieval step
Heat mediated
Permeabilization
Yes - Tween-20
Specification
fetal spinal cord nerve fibers
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde

Mr. Heiko Locher

Verified customer

Submitted Sep 23 2013

Answer

Thank you very much for your reply, and I am very sorry for all of the trouble with these antibodies.

Your credit note ID is ***

If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used.

Our accounting department can be contacted by email at us.credits@abcam.com or by telephone at 888-772-2226. Please refer to the credit note ID in any correspondence with our accounting department.

Please let me know if you have any questions or if there is anything else that we can do for you.

Read More

Answer

Thank you for sending the additional information and for your patience.

I forwarded your protocol details to the lab for a second look, and we would have expected the antibody to work in the conditions that you've described. I am very sorry that neither of these antibodies has worked for you, and I can arrange a credit or a refund for your original purchase if you'd like. Alternatively, we do have one other antibody that is predicted to work with rat samples, ab135457-

https://www.abcam.com/Nav16-antibody-ab135457.html

This antibody hasn't been tested with rat samples, but since the immunogen is homologous to the rat protein it may work. I'd be happy to send this out if you'd like to try it.

I apologize again for the poor results with these Nav1.6 antibodies, but if there is anything else that we can do for you, please let me know.

Read More

Answer

Thank you very much for keeping me updated about the results with ab65166.

I'm sorry to hear that there was still no band on the blot. Would you mind telling me some more about the protocol you used with ab65166?

1) Are you still using rat hippocampus lysates? How much protein was loaded per lane of the gel?

2) Could you describe the electrophoresis and gel conditions (% of the gel etc.)?

3) Could you describe the transfer and blocking conditions (buffer, transfer time, blocking agent etc.)?

4) Does the secondary antibody work with other primaries?

5) Detection method (ECL, ECLPlus etc.)

6) Did the ladder proteins transfer, or did you confirm transfer of the high molecular proteins via Ponceau stain?

We don't have any other antibodies to Nav1.6 that have been tested in Western blot with rat lysates, but I'll see if there are any protocol suggestions that we can make. If the results are still not satisfactory, we can arrange a credit or refund for your original purchase.

Please let me know if you have further questions or comments, and I'll be happy to help.

Read More

Question
Answer

Thank you very much for your calls today and for letting us know about the trouble with ab83764 in Western blot.

Please keep me updated about the results after using ab65166 with your samples.

I look forward to hearing from you. If you have any questions or if there is anything else that we can do for you, please let me know and I'll be happy to help. Have a nice weekend!

Read More

Answer

I am sorry to hear that you have been experiencing problems using this product in the application that you wish.

In order to assess the quality of our products I would ask that you complete a brief questionnaire relating to the application used. Often it is possible to make suggestions that may help resolve problems experienced using a particular product.

As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

All our customer feedback, including complaints are monitored weekly by our in house technical support team. If a product is at fault the technical support team will consider removing the product from our catalogue in order to avoid future customer inconvenience.

Could you please provide some further details of the protocol used and complete the following form (attached as a word document). It would be much appreciated if you could attach an image to the response.

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.

Read More

Question

Thank you for your helpful advice. I used Ponceau S on my most
recent blot and was able to visualize transfer of both my MW marker
and proteins in my cell lysate. It's certainly possible, however,
I could be getting over-transfer to some extent. I recently contacted
the lab that wrote the review for ab65166 on your website, and they
said that they performed a 16 h transfer at 20 V. With the new lysate,
my hope is to try both your one hour transfer conditions and the 16 h
transfer conditions and see if I see better transfer with either.
I'd also be open to trying higher MeOH and lower SDS content;
although, the aforementioned group apparently used a basic transfer
buffer with 20% MeOH and no SDS.
I'm not currently blotting for a loading control, but that is
a good suggestion. The best evidence I have for the success of my
WB protocol is that I could visualize Invitrogen's Precision
Plus MW marker after incubation with my secondary antibody.
In terms of lysing my own sample, I used the following reference to
prepare my lysis buffer (http://www.springerprotocols.com/Full/doi/
10.1007/978-1-61779-328-8_23?encCode=U0VOOjMyXzgtODIzLTk3NzE2LTEtODc5
&tokenString=k/NnQ1wdXIf1ihPjyV6QSQ==).
I certainly am open to try other lysis buffers but felt it might
be best to use a formulation that had been used successfully with
these channels before.
I very much appreciate the offer to send me another vial of ab30151.
From some initial reading, it looks like Nav1.6 expression increases
during maturation, so it's perhaps best to stick with the lysate
I'm already using. The order number for the original order is
xxx.
I can't thank you all enough for your help so far. Your team
has been incredibly responsive, and I very much appreciate your
assistance.

Read More
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 with the order number xxx. The estimated delivery date is xxx as the item is currently backordered. I hope this is not causing a big problem. I apologize.

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

Thank you for your suggestions. In reviewing my previous blots, I
realized that all the ones I had run with the commercial cell lysate
had been stained with Amido Black, which made them unsuitable for
stripping and re-probing. I consequently ran another gel, loading
15 ug of ab30151 as well as the lysate for the system I'm trying
to study. I ran my transfer similar to before (75 V for 4 hours),
using 5% MeOH and 0.5% SDS in my transfer buffer, similar to your
suggestion. After transfer, I can see my MW marker (other company) fully transferred. I then pre-blocked with 3% BSA before
incubating with my primary antibody (ab65166) in 3% BSA overnight
at 4 oC. Finally, I incubated with the secondary antibody in 3%
BSA for 1 hour at room temperature and then did 3 10 minutes washes
with TBS-Tween before a final wash with TBS.
Upon visualization with my ECL+ kit, I only saw very high background
at moderate exposure times (1 minute) and couldn't see any signal
above background at very short exposure times (5-10 seconds). In
hindsight, I believe I should have incubated with the secondary
antibody in 10% milk.
I tried stripping the blot using the mild stripping protocol on
your website but still saw an almost equally strong background
upon visualization. At this point, I assume I must still have a
lot of secondary antibody non-specifically bound to the membrane,
so my next step is to try the harsh conditions.
Even though I see my full MW ladder transferred, I could definitely
believe that my transfer is still sub-optimal. I would like to try
alternative transfer conditions, but I have very little of my
remaining lot of ab30151 left. Would it be possible for me to obtain
another lot of this lysate at a discounted price? Alternatively, is
there another item in your catalog that you would recommend as a
positive control for the primary antibody that I could pay full price
for?

Read More
Answer

Thank you for your reply.


I have a few additional suggestions. I am not sure if my colleague had mentioned those to you:

1) You can check the transfer with Ponceau S (which is a reversible membrane stain) to be sure that you transfered the proteins. This will give you also an additonal quality control for the transfer. My feeling is that transferingat 75V for 4 h could be too long. if the gel can withstand a higher voltage, I'd suggest to try 100V for an 1 h at 4 degree with stirring.

2) Further, I'd suggest to use 10% MeOH and reduce the amount of SDS to 0.1% in the transfer buffer. You want to have some SDS to prevent hydrophobic proteins (like Nav1.6) from precipitating in the gel during the transfer, but 0.5% seems a bit high.

3) Are you blotting for any other proteins, such as actin or tubulin as loading controls? This would be another way to check if the WB protocol in general works and if the mouse brain lysate you have is OK.

4) How are your samples lysed? I would recommend using RIPA buffer since the Nav1.6 protein is very hydrophobic.

I also checked online and found the following link for WB with membrane proteins:
http://www.sciencedirect.com/science/article/pii/S0003269708006829

Our WB protocol guide can be found here: https://www.abcam.com/index.html?pageconfig=resource&rid=11375and I have it also attached to this email.

To answer your question about the lysate:

I'd be happy to send you another vial of ab30151 free of charge. Alternatively, we also have ab7188 (Brain (Mouse) Tissue Lysate - normal tissue, 0 days old) [https://www.abcam.com/Brain-Mouse-Tissue-Lysate-normal-tissue-0-days-old-ab7188.html] in the catalog, but I am not certain if newborn mice express this protein strongly.
I can send you 1 vial of either one or the other free of charge. Please let me know which one you would prefer. Please let me know your order or PO number for the lysate.

I look forward to hear back from you and assist you further.

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Read More

Question
Answer

Thank you for calling and bringing this issue to our attention.

I just wanted to make sure you were loading the correct amount of protein and to make another suggestion regarding the blocking step.

For the brain lysate, ab30151, you will want to load at least 10ug, which is a volume of about 4 microliters.

For the blocking step, you may find that a relatively short block (30 minutes) instead of overnight, followed by overnight incubation with the antibody will help develop the signal, compared to an overnight block followed by a shorter incubation with the primary. These are little modifications, though. I suspect that either the lysate is defective or the transfer was not optimal. Please let me know how your tests go. If they fail, we will be happy to discuss a replacement of either the antibody or the lysate, or both.

Read More
Application
Western blot
Sample
Mouse Tissue lysate - whole (Whole brain homogenate)
Loading amount
10 µg
Specification
Whole brain homogenate
Gel Running Conditions
Reduced Denaturing (Bis-tris 4-12%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Mr. Brian Hitt

Verified customer

Submitted May 11 2010

1-10 of 12 Abreviews or Q&A

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