Overview

  • Product nameAnti-MiTF antibody [D5]See all MiTF primary antibodies ...
  • Description
    Mouse monoclonal [D5] to MiTF
  • Specificityab3201 does not cross-react with other b-HLH-ZIP factors by DNA mobility shift assay. It reacts with both melanocytic and non-melanocytic isoforms of Mi. It is especially useful for research on murine Mi protein.
  • Tested applicationsICC/IF, WB, IHC-P, Flow Cyt, Gel supershift assaysmore details
  • Species reactivity
    Reacts with: Dog, Human
    Predicted to work with: Mouse

    Does not react with

    Rat
  • Immunogen

    N-terminal fragment of human Mi protein.

  • EpitopeN-terminal
  • Positive control
    • 501 Mel cells Melanoma
      In Flow Cytometry, this antibody gave a positive signal in methanol fixed/Tween permeabilised Malme-3M cells.
  • General notesWe can conjugate this antibody to FITC for you (please see ab150228 for details).

Properties

Applications

Our Abpromise guarantee covers the use of ab3201 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 at an assay dependent concentration.
WB Use at an assay dependent concentration. Predicted molecular weight: 59 kDa.
IHC-P Use at an assay dependent concentration.
Flow Cyt Use 1µg for 106 cells.
Gel supershift assays Use at an assay dependent concentration.

Use at an assay dependent concentration

Target

  • FunctionTranscription factor for tyrosinase and tyrosinase-related protein 1. Binds to a symmetrical DNA sequence (E-boxes) (5'-CACGTG-3') found in the tyrosinase promoter. Plays a critical role in the differentiation of various cell types as neural crest-derived melanocytes, mast cells, osteoclasts and optic cup-derived retinal pigment epithelium.
  • Tissue specificityIsoform M is exclusively expressed in melanocytes and melanoma cells. Isoform A and isoform H are widely expressed in many cell types including melanocytes and retinal pigment epithelium (RPE). Isoform C is expressed in many cell types including RPE but not in melanocyte-lineage cells.
  • Involvement in diseaseDefects in MITF are the cause of Waardenburg syndrome type 2A (WS2A) [MIM:193510]. It is a dominant inherited disorder characterized by sensorineural hearing loss and patches of depigmentation. The features show variable expression and penetrance.
    Defects in MITF are a cause of Waardenburg syndrome type 2 with ocular albinism (WS2-OA) [MIM:103470]. It is an ocular albinism with sensorineural deafness.
    Defects in MITF are the cause of Tietz syndrome (TIETZS) [MIM:103500]. It is an autosomal dominant disorder characterized by generalized hypopigmentation and profound, congenital, bilateral deafness. Penetrance is complete.
  • Sequence similaritiesBelongs to the MiT/TFE family.
    Contains 1 basic helix-loop-helix (bHLH) domain.
  • Post-translational
    modifications
    Phosphorylation at Ser-405 significantly enhances the ability to bind the tyrosinase promoter.
  • Cellular localizationNucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • BHLHE32 antibody
    • bHLHe32 antibody
    • Class E basic helix-loop-helix protein 32 antibody
    • CMM8 antibody
    • Homolog of mouse microphthalmia antibody
    • Mi antibody
    • Microphthalmia associated transcription factor antibody
    • Microphthalmia, mouse, homolog of antibody
    • Microphthalmia-associated transcription factor antibody
    • MITF antibody
    • MITF_HUMAN antibody
    • WS2 antibody
    • WS2A antibody
    see all

Anti-MiTF antibody [D5] images

  • All lanes : Anti-MiTF antibody [D5] (ab3201) at 5 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 59 kDa
    Observed band size : 40 kDa (why is the actual band size different from the predicted?)


    Exposure time : 10 minutes
  • Overlay histogram showing Malme-3 cells stained with ab3201 (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. The cells were then incubated with the antibody (ab3201, 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.

  • ab3201 staining MiTF in the SK-MEL-28 (a human melanoma cell line) by ICC/IF(Immunocytochemistry/immunofluorescence). Cells were fixed with Paraformaldehyde and permeabilized with Triton X-100 0.5% in PBS. Samples were incubated with primary antibody (1/100) for 24 hours at 4°C. An Alexa Fluor®488-conjugated Goat anti-mouse IgG polyclonal(1/1000) was used as the secondary antibody. SK-MEL-28 cells was grown on chamber slide. MiTF was found to strictly localised in the nucleus. Cells were counter-stained with DAPI.

    See Abreview

References for Anti-MiTF antibody [D5] (ab3201)

This product has been referenced in:
  • Zahabi A  et al. A New Efficient Protocol for Directed Differentiation of Retinal Pigmented Epithelial Cells from Normal and Retinal Disease Induced Pluripotent Stem Cells. Stem Cells Dev : (2012). Read more (PubMed: 22145677) »
  • Petersen-Jones SM  et al. Ocular melanosis in the Cairn Terrier: histopathological description of the condition, and immunohistological and ultrastructural characterization of the characteristic pigment-laden cells. Vet Ophthalmol 11:260-8 (2008). IHC-P ; Dog . Read more (PubMed: 18638353) »

See all 2 Publications for this product

Product Wall

Thank you for contacting Abcam yesterday.

I am working with our lab to try and understand where the data stating there was a lack of reactivity with mouse came from, as we have not tested it on mouse. I do apologize for the confusion and for ...

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Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (SK-MEL-28 (human melanoma cell line))
Specification SK-MEL-28 (human melanoma cell line)
Fixative Paraformaldehyde
Permeabilization Yes - 0.5% Triton-X in PBS
Username

Abcam user community

Verified customer

Submitted Oct 16 2012

Thank you for your reply.

I can confirm that this antibody has been tested successfully in WB with blocking in 5% nonfat dry milk with PBS + 0.1%Tween 20. However, please note this may require some individual optimization.

If you ...

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Thank you for your enquiry.

I would be pleased to help answer your questions. I assume you are using the western blot application?

Regarding dilution to use the antibody in WB, the details on the online datasheet for ab79475 recom...

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Thank you for your email. I'm happy to hear that the new batch is working for your. I was able to obtain an image of human melanoma stained with ab3201 which I'm sending you as a separate email. Please let me know if you have any additional questions.

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Thank you for your email. I unfortunately cannot send you a positive control free of charge. 501 Mel cells and Melanoma are recommended as positive controls for ab3201. If you have any additional questions, please let us know.

Thank you for your email. I contacted the originator of this antibody concerning the problem that you were experiencing. It turns out that there is a problem with that particular batch and so I'm sending you a vial from a new batch free of charge. This...

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Thank you for your reply and for providing more information about your order details. This is to let you know that we have placed a new order for you - one vial of ab3201 free of charge. The new order number is 56046. Please do let us know how ...

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Unfortunately we do not have the sequence for this antibody, sorry for the incovenience and the delay.

Thank you for contacting us and providing more details of your protocol. We are very sorry to hear that you are having problem with this antibody. We have retested this antibody and would like to make the following suggestions: 1. For formalin-fi...

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"