Recombinant
RabMAb

Recombinant Anti-TMEM16A antibody [SP31] (ab64085)

Rabbit monoclonal TMEM16A antibody [SP31]. Validated in WB, IHC, ICC/IF and tested in Mouse, Human, Cynomolgus monkey. Cited in 26 publication(s). Independently reviewed in 1 review(s).

Overview

  • Product name

    Anti-TMEM16A antibody [SP31]
    See all TMEM16A primary antibodies
  • Description

    Rabbit monoclonal [SP31] to TMEM16A
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IHC-P, Flow Cyt, ICC/IF, WBmore details
  • Species reactivity

    Reacts with: Mouse, Human, Cynomolgus monkey
  • Immunogen

    Synthetic peptide within Human TMEM16A aa 400-500. The exact sequence is proprietary.
    Database link: Q5XXA6

  • Positive control

    • Human GIST tumor FC: PC-3 cells.
  • General notes

    ab64085 was switched from a hybridoma to recombinant production method on 23rd July 2019.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • High batch-to-batch consistency and reproducibility
    • Improved sensitivity and specificity
    • Long-term security of supply
    • Animal-free production
    For more information see here.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
  • Storage buffer

    pH: 7.6
    Preservative: 0.1% Sodium azide
    Constituents: 1% BSA, PBS
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    SP31
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab64085 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-P 1/100.
Flow Cyt Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration. PubMed: 23866066
WB Use at an assay dependent concentration. Predicted molecular weight: 114 kDa. PubMed: 26121472

Target

  • Function

    Acts as a calcium-activated chloride channel. Required for normal tracheal development.
  • Tissue specificity

    Broadly expressed with higher levels in liver and skeletal muscle.
  • Sequence similarities

    Belongs to the anoctamin family.
  • Domain

    The region spanning the fifth and sixth transmembrane domains probably forms the pore-forming region.
  • Cellular localization

    Cell membrane. Cytoplasm.
  • Information by UniProt
  • Database links

  • Alternative names

    • ANO 1 antibody
    • ANO1 antibody
    • ANO1_HUMAN antibody
    • Anoctamin 1 antibody
    • Anoctamin 1 calcium activated chloride channel antibody
    • Anoctamin-1 antibody
    • Anoctamin1 antibody
    • Ca2+ activated Cl- channel antibody
    • Calcium Activated Chloride Channel antibody
    • Discovered on gastrointestinal stromal tumors protein 1 antibody
    • DOG 1 antibody
    • DOG1 antibody
    • FLJ10261 antibody
    • Membrane protein antibody
    • Oral cancer overexpressed 2 antibody
    • Oral cancer overexpressed protein 2 antibody
    • ORAOV 2 antibody
    • ORAOV2 antibody
    • TAOS 2 antibody
    • TAOS2 antibody
    • TMEM 16A antibody
    • TMEM16A antibody
    • Transmembrane protein 16A (eight membrane spanning domains) antibody
    • Transmembrane protein 16A antibody
    • Tumor amplified and overexpressed sequence 2 antibody
    • Tumor-amplified and overexpressed sequence 2 antibody
    see all

Images

  • Flow Cytometry analysis of PC-3 (Human prostate adenocarcinoma epithelial cell) cells labeling TMEM16A with purified ab64085. (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / blue.

  • Western blot analysis of TMEM16A protein expression useing ab64085. Non-CF cells were treated with pyocyanin (60 μM) or IL-4 (10 ng/ml) for 24 hours. Pyocyanin and, to a larger extent, IL-4 increased TMEM16A expression. Each panel is representative of three similar experiments.

  • Representative images showing detection of TMEM16A with ab64085 (green), MUC5AC (red), and acetylated tubulin (magenta) by immunofluorescence. Images, taken with a confocal microscope, are xy (top) and xz (bottom) scans of cultured bronchial epithelial cells treated with and without pyocyanin. The xz images also report staining of nuclei with DAPI (blue). The blue layer under the cells is the porous membrane of the Snapwell insert.

    https://doi.org/10.1371/journal.pone.0131775.g004

  • TMEM16A detected in paraffin-embedded  sections of human submucosal glands from CF patients and control samples.

    TMEM16A expression was modest in non-CF submucosal glands of non-CF samples (b) but markedly increased in tissues from CF patients (d), with a particularly strong signal (f) in histologically altered glands (e). Magnification: X630 in a, X400 in b-f. Images 4Aa and 4Ba,c,e show hematoxylin and eosin staining.

  • ab64085 showing expression of TMEM16A in human surface epithelium tissue. TMEM16A staining was mostly absent (b), and sometimes scanty (c) in the respiratory epithelium lining bronchi or bronchioles from CF patients (c, arrows). A weak expression was also detectable in microvessels of peri-bronchial connective tissue (b, arrows). Magnification: X400.

  • Immunohistochemical analysis of Human Gastrointestinal Stromal Tumor tissue labelling TMEM16A with ab64085.

References

This product has been referenced in:

  • Miner K  et al. Drug Repurposing: The Anthelmintics Niclosamide and Nitazoxanide Are Potent TMEM16A Antagonists That Fully Bronchodilate Airways. Front Pharmacol 10:51 (2019). Read more (PubMed: 30837866) »
  • Zhou Y  et al. Age-associated variation in the expression and function of TMEM16A calcium-activated chloride channels in the cochlear stria vascularis of guinea pigs. Mol Med Rep 20:1593-1604 (2019). Read more (PubMed: 31257512) »
See all 31 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Answer

I have had the opportunity to review our anti-TMEM16A antibodies and have found two which will recognize the extracelluar domain of this target. https://www.abcam.com/tmem16a-antibody-ab84115.html was created from a peptide corresponding to a region within amino acids 828-877 (HGFVNHTLSS FNVSDFQNGT APNDPLDLGY EVQICRYKDY REPPWSENKY) of Human TMEM16A (NP_060513). Based on Uniprot information (Q5XXA6 ) this region is extracellular. https://www.abcam.com/tmem16a-antibody-sp31-ab64085.html was made using a cocktail of 3 different immunogens; two are from the cytoplasmic domain and one is from the extracellular domain.

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Question

Hi there,

Please find below the completed form.

Thanks for your support.

Sincerely.




1) Abcam product code ab 64085

2) Abcam order reference number or product batch number
3) Description of the problem no specific signal

4) Sample preparation:
Species mouse gut
Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other: overnight fixation in 4% paraformaldehyde followed by graded sucrose solutions (10-20-30%), OCT embedding and cryostat sectioning.
Sample preparation
Positive control : yes, another rabbit Ab labeling the same cell type
Negative control : yes – omission of primary Ab

5) Fixation step
Yes/No
If yes: Fixative agent and concentration paraformaldehyde 4%
Fixation time overnight
Fixation temperature 4°C

6) Antigen retrieval method : no

7) Permeabilization method:
Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers?
Permeabilizing agent and concentration: TritonX 0.1% diluted with the blocking agent and the primary antibody


8) Blocking agent (eg BSA, serum…): normal horse serum
Concentration 10%
Blocking time 60 minutes
Blocking temperature RT

9) Endogenous peroxidases blocked? no
Endogenous biotins blocked?

10) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution 1/100 - 1/500 - 1/1000
Diluent buffer TBS 0.01M + TRITON x 0.1%
Incubation time overnight at RT

11) Secondary antibody:
Species: donkey
Reacts against: rabbit IgG
Concentration or dilution 1/200
Diluent buffer TBS 0.01M pH8.2
Incubation time 1H
Fluorochrome or enzyme conjugate: biotinylated secondary Ab + streptavidin˜Dyelight647 or secondary Ab coupled to Cy3

12) Washing after primary and secondary antibodies:
Buffer TBS 0.01M pH8.2
Number of washes 3x5min

13) Detection method fluorescence microscopy

14) How many times have you run this staining? two times
Do you obtain the same results every time? yes
What steps have you altered to try and optimize the use of this antibody? various dilutions, several different samples from different animals, differnt secondary Abs

Document attachment: no image to attach.

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Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

I would like to confirm that this antibody was not yet tested in IHC-Fr.

In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Reviewing this case, I would like to offer some suggestions to help optimise the results from ab64085.

1.) TMEM16A is a multi pass membrane protein. Triton X is a harsh detergent and will disrupt proteins when used for longer amounts of time, especially affection results of membrane proteins. We in general suggest only 10 minutes incubation with 0.1% Triton X.

I suggest to use tween 20, Saponin, Digitonin or Leucoperm instead.

These are much milder membrane solubilizers. They will give large enough pores for antibodies to go through without dissolving the plasma membrane. They are suitable for antigens in the cytoplasm or the cytoplasmic face of the plasma membrane and for soluble nuclear antigens.

2.) Fixing in paraformaldehyde for more than 10-15 min will cross link the proteins to the point where antigen retrieval may be required to ensure the antibody has free access to bind and detect the protein. Maybe Acetone will improve the results since it also permeabilises.

3.) Consider including 0.3 M glycine in the blocking buffer, before applying the primary antibody. Glycine will bind free aldehyde groups that would otherwise bind the primary and secondary antibodies, leading to high background. Background due to free aldehyde groups is more likely to occur when the fixative is glutaraldehyde or paraformaldehyde.

I would also appreciate if you can confirm some further details:

1.) What was the positive control?

2.) Which species and tissues were used?

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Read More

Answer

Thank you for your reply and for kindly providing the word document. This has made conducting the alignments much more straight forward.

I am sorry to confirm that as far as we are aware,none of our TMEM16A antibodieshas been tested with samples from Zebrafish. All tested species covered by the 6 month guarantee are stated on our datasheets, and these are updated as soon as any new information is brought to our attention.

I have obtained the immungoen sequences for all our TMEM16A antibodies and checked the alignment of these with the 3 Zebrafish sequences you provided.

Regrettably, for most of these the alignments are too low for us to suggest they may detect in Zebrafish. However, there is one with reasonable alignment that you may like to consider trying. I can recmomend to review the online datasheet for further information.

ab84115
https://www.abcam.com/index.html?datasheet=84115 (or use the following: https://www.abcam.com/index.html?datasheet=84115).

Immunogen: ################

Alignment with Zebrafish sequences provided:
Sequence 1: 68%
Sequence 2: 68%
Sequence 3: 72%

We recommend alignment should be over 85% to predict that an antibody will detect. It isstill possible that with 68-72% alignment therecould be some crossreactivity. However, even when alignmentis very highwe cannot guarantee this without further testing.


If you would like to test the ab84115 in Zebrafish, please contact me again by replying to this message prior to the purchase as you may be eligible for our testing discount program.

Otherwise, we like to encourage all of our customers to submit an Abreview via the online product datasheet. We always appreciate customer feedback, whether positive or negative, and we make all product information available to researchers. Each Abreview earns Abpoints that can be used for discounts on future purchases or rewards such as Amazon.com gift certificates.

To find out more about our Abreview system, please see the following link:

https://www.abcam.com/abreviews

I hope this information is helpful. Please do not hesitate to contact me for any further advice or information.

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Question
Answer

Thank you for your phone call.

DISCOUNT CODE: xxx
Expiration date: xxx

I am very pleased to hear you would like to accept our offer and test ab64085 in ICC/IF. This code will give you: 1 free PRIMARY ANTIBODY before the expiration date. To redeem this offer, please submit an Abreview for ICC/IFand include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.
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: https://www.abcam.com/collaborationdiscount.

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Question
Answer

Thank you for contacting Abcam regarding ab64085.


The immunogen for this antibody is not located within aa 650-706 thus it should not be affected by the paper's findings (Circ Res March 30 2012. Vol 110, p990-999 ).

Please do not hesitate to contact me with additional questions or concerns.

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Question
Answer

ab64085 was made using a cocktail of 3 different immunogens; one is from the pore-forming region and the other two are not.  Unfortunately the epitope for this antibody has not been mapped, so we do not know where the target is.  Please do not hesitate to contact me with additional questions.

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Answer

Unfortunately the exact immunogen sequence of this antibody is proprietary. I can tell you that the Anti-TMEM16A antibody [SP31] (ab64085) which you are interested is produced using a cocktail of 3 peptides, two of which are located in cytoplasmic and one of which is in the extracellular domain of TMEM16A. I hope this information helps, if you have any further questions please do not hesitate to ask.

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Application
Western blot
Sample
Rat Tissue lysate - whole (Whole brain)
Gel Running Conditions
Reduced Denaturing (7.5)
Loading amount
40 µg
Treatment
1X SDS for 10 mins
Specification
Whole brain
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

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Submitted Nov 06 2009

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