Anti-MiTF antibody [D5] (ab3201)

Reviews (1)Q&A (10)References (8)

Overview

  • Product name
    Anti-MiTF antibody [D5]
    See all MiTF primary antibodies
  • Description
    Mouse monoclonal [D5] to MiTF
  • Host species
    Mouse
  • Specificity
    ab3201 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 applications
    Suitable for: ICC/IF, WB, IHC-P, Flow Cytmore details
  • Species reactivity
    Reacts with: Dog, Human
    Predicted to work with: MouseDoes not react with: Rat
  • Immunogen

    N-terminal fragment of human Mi protein.

  • Epitope
    N-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 notes

    This antibody clone 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 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
EMSA Use at an assay dependent concentration.

Use at an assay dependent concentration
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. PubMed: 18638353
Flow Cyt Use 1µg for 106 cells.

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

 

Target

  • Function
    Transcription 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 specificity
    Isoform 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 disease
    Defects 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 similarities
    Belongs 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 localization
    Nucleus.
  • Information by UniProt
  • Database links
    see all
  • 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
    • mitfa antibody
    • nacre antibody
    • WS2 antibody
    • WS2A antibody
    • z3A.1 antibody
    see all

Images

  • Western blot - Anti-MiTF antibody [D5] (ab3201)
    Western blot - Anti-MiTF antibody [D5] (ab3201)
    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
    All lanes : 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
  • Immunocytochemistry/ Immunofluorescence - Anti-MiTF antibody [D5] (ab3201)
    Immunocytochemistry/ Immunofluorescence - Anti-MiTF antibody [D5] (ab3201)This image is courtesy of an anonymous Abreview
    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

  • Immunocytochemistry/ Immunofluorescence - Anti-MiTF antibody [D5] (ab3201)
    Immunocytochemistry/ Immunofluorescence - Anti-MiTF antibody [D5] (ab3201)

    ICC/IF image of ab3201 stained Malme-3M cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab3201 at 5µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- mouse (ab150117) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

  • Flow Cytometry - Anti-MiTF antibody [D5] (ab3201)
    Flow Cytometry - Anti-MiTF antibody [D5] (ab3201)
    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.

References

This product has been referenced in:
  • Fernandes M  et al. Stem Cell-Derived Retinal Pigment Epithelial Layer Model from Adult Human Globes Donated for Corneal Transplants. Curr Protoc Stem Cell Biol 45:e53 (2018). Read more (PubMed: 30040247) »
  • Saini JS  et al. Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-Related Macular Degeneration. Cell Stem Cell : (2017). Read more (PubMed: 28132833) »
See all 8 Publications for this product

Publishing research using ab3201? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-10 of 11 Abreviews or Q&A

Question

Product code: 3201
Lot number: GR44826-1
Inquiry: A few questions regarding anti MITF ab 3201: - Should it be incubated with BSA or dried milk in TBST, and at what concentration? - What is the recomended incubation time- and temperature? (If you could also supply the same information about Anti- MAGE-1 antibody MA454 ab 79475?) Best regards

Read More
Answer

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 recommend tousethe antibody at aconcentration of 0.5 - 1 ug/ml. For ab3201, the image on the datasheet includes information that this has been used at 5 ug/ml. Please note that these concentrations area guideline only and may require some individual optimization.

The incubation time and temperature will also require some individual optimization. I would recommend to try 4oC overnight. This can often provide the most efficient and specific staining.

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

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Question

How was this antibody determined not react with mouse. Customer has antibody and is working really well in IF on mouse samples?

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Answer

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 bringing this to our attention. Once we have looked into this more thoroughly, we will update the datasheet as necessary.

If there is anything else I can help you with, please let me know.

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Immunocytochemistry/ Immunofluorescence abreview for Anti-MiTF antibody [D5]

 Good
Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
abreview image
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
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Abcam user community

Verified customer

Submitted Oct 16 2012

Question

Hi!
Thank you for your reply,
I would also very much like to know if the antibody works best with
TBS-T w/dry milk or BSA.
Best regards

Read More
Answer

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 have any further questions, please do not hesitate to contact us.

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Question

I tested the new antibody that AbCam sent me, and it worked. The staining (IHC), however, was not as strong as I would have imagined, even though we were using a 1:4 dilution. Do you have any pictures of positive control slides stained with MiTF that I could compare my results to?

Read More
Answer

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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Question

Could I also get positive control slides from you so that I can test to see whether the new antibody is good?

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Answer

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.

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Question

BATCH NUMBER Lot# 761 ORDER NUMBER 022T0022 DESCRIPTION OF THE PROBLEM No staining when doing immunohistochemistry with positive control (melanoma). Tried 8 different dilutions ranging from 1:2 to 1:100. Would like to receive a new batch of antibody with positive control slides. The test run was done with other antibodies on different slides, and all antibodies but MITF worked as expected. Used Dako Envision kit and citrate buffer at pH 6 for antigen retrieval. HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes

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Answer

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 is on order# 79431 and will take approximately 1 week for you to receive it as we have to first receive it from the originator. You will receive email updates regarding shipping. Also, you previously contacted me back in March because the vial that you received was labeled [C5] instead of [D5]. I had thought that there was a mis-labeling problem but the clone number was changed from [C5] to [D5] this past September. If you have any additional questions, please do not hesitate to contact us again.

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Question

I have been using this antibody for more than a (2)years. The procedure that I sent you has not changed. I subsequently have ordered more of the same antibody and have good results. I don't need to change my procedure. I got a bad lot of antibody and all I want is a replacement in a timely order and the case will be closed. Procedure: Formalin fixed tissue, paraffin embedded tissue , dog tissue, run on an automated system: Ventana's BenchMark, 1:100 @32 min antibody incubation, using detection kit: BMK Enhanced V-Red Par Plus, antigen retrieval is "standard" duration with CC1, 2 min hematoxylin , 2 min Bluing Reagent. Dehydrate with 95% then 100% (6) dips each, cleared with (3) changes of Xylene (6) dips each then immediately cover slipped with an aqueous mounting medium. FYI: I had just enough of the previous lot# to run a few slides with controls and they worked perfectly. The only variable is the new lot#.

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Answer

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 you are getting on.

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Question

Could you let me know sequence of immunogen of MiTF antibody [D5] (ab3201)? Thank you for helping,

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Answer

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

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Question

DESCRIPTION OF THE PROBLEM No staining or very very poor staining SAMPLE Canine PRIMARY ANTIBODY ab3201-500 1:100 SECONDARY ANTIBODY Ventana Enhanced Vetor Red Par plus kit DETECTION METHOD Bench Mark POSITIVE AND NEGATIVE CONTROLS USED Positive MiTF ANTIBODY STORAGE CONDITIONS 4 degrees Celcius in original vial FIXATION OF SAMPLE Formalin ANTIGEN RETRIEVAL HEIR HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Protocal is identical to previous successful positive runs with older antibody of identical cat number. ADDITIONAL NOTES The only variable is the newly purchased antibody C5 to MiTF (cat#3201-500 , lot#58895)

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Answer

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-fixed sections, try to incubate the samples wit this primary antibody at 4-8 ug/ml concentration for 30 min at room temperature. 2. Staining of formalin-fixed tissue requires boiling tissue sections in 10 mM citrate buffer (pH 6.0) for 10-20 min followed by cooling at RT for 20 min. 3. Use recommended positive control - human melanoma. We hope this will help. Should you still need further assistance, then please do not hesitate to contact us again.

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