Anti-MiTF antibody [C5] (ab12039)

Reviews (5)Q&A (10)References (63)

Key features and details

  • Mouse monoclonal [C5] to MiTF
  • Suitable for: IHC-Fr, IHC-P, ICC/IF, Flow Cyt, WB, IP, GSA
  • Reacts with: Mouse, Rat, Chicken, Human
  • Isotype: IgG1

Overview

  • Product name

    Anti-MiTF antibody [C5]
    See all MiTF primary antibodies

  • Description

    Mouse monoclonal [C5] to MiTF

  • Host species

    Mouse

  • Tested applications

    Suitable for: IHC-Fr, IHC-P, ICC/IF, Flow Cyt, WB, IP, GSAmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Chicken, Human

  • Immunogen

    Recombinant fragment corresponding to Human MiTF (N terminal).

  • Positive control

    • WB: Malme-3M whole cell lysate. Flow Cyt: 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 ab12039 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-Fr Use at an assay dependent concentration.
IHC-P Use a concentration of 1 µg/ml. Antigen retrieval is not essential but may optimise staining.
ICC/IF Use at an assay dependent concentration. PubMed: 16648630

Fix with methanol/acetone, block with 1% BSA/PBS (see Wellbrock et al).
EMSA Use at an assay dependent concentration.
Flow Cyt Use 2µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 52-56 kDa (predicted molecular weight: 59 kDa).
IP Use at 2 µg/mg of lysate.
GSA Use at an assay dependent concentration.

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 [C5] (ab12039)
    Western blot - Anti-MiTF antibody [C5] (ab12039)
    Anti-MiTF antibody [C5] (ab12039) at 1 µg/ml + Malme 3m (Human melanoma cells) Whole Cell Lysate at 10 µg

    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: 40,55,70 kDa
    why is the actual band size different from the predicted?


    Exposure time: 20 minutes
  • Immunohistochemistry (Frozen sections) - Anti-MiTF antibody [C5] (ab12039)
    Immunohistochemistry (Frozen sections) - Anti-MiTF antibody [C5] (ab12039)Image courtesy of Katia Del Rio-Tsomis by Abreview.
    ab12039 staining MiTF in embryonic day 4 chick eye tissue by Immunohistochemistry (Frozen sections).
    Tissue was fixed with 4% paraformaldehyde, permeabilized using 1% saponin, blocked with 10% serum for 30 minutes at 37°C, then incubated with ab12039 at a 1/1000 dilution for 24 hours at 4deg;C. The secondary used was an Alexa-Fluor 488 conjugated mouse monoclonal, used at a 1/100 dilution. Counterstained with DAPI (blue).

    See Abreview

  • Flow Cytometry - Anti-MiTF antibody [C5] (ab12039)
    Flow Cytometry - Anti-MiTF antibody [C5] (ab12039)
    Overlay histogram showing Malme 3m cells stained with ab12039 (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 (ab12039, 2µg/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

Publishing research using ab12039? Please let us know so that we can cite the reference in this datasheet.

ab12039 has been referenced in 63 publications.

  • Pogenberg V  et al. Mechanism of conditional partner selectivity in MITF/TFE family transcription factors with a conserved coiled coil stammer motif. Nucleic Acids Res 48:934-948 (2020). PubMed: 31777941
  • Yun CY  et al. Stem Cell Factor-Inducible MITF-M Expression in Therapeutics for Acquired Skin Hyperpigmentation. Theranostics 10:340-352 (2020). PubMed: 31903124
  • Gaudel C  et al. Regulation of Melanogenesis by the Amino Acid Transporter SLC7A5. J Invest Dermatol N/A:N/A (2020). PubMed: 32240722
  • Jiang L  et al. Ganoderma lucidum polysaccharide reduces melanogenesis by inhibiting the paracrine effects of keratinocytes and fibroblasts via IL-6/STAT3/FGF2 pathway. J Cell Physiol 234:22799-22808 (2019). PubMed: 31115052
  • Osmanbeyoglu HU  et al. Chromatin-informed inference of transcriptional programs in gynecologic and basal breast cancers. Nat Commun 10:4369 (2019). PubMed: 31554806
View all Publications for this product

Customer reviews and Q&As

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1-10 of 15 Abreviews or Q&A

Immunohistochemistry (Frozen sections) abreview for Anti-MiTF antibody [C5] - ChIP Grade

 Good
Abreviews
abreview image
Application
Immunohistochemistry (Frozen sections)
Sample
Chicken Tissue sections (Cryostat tissue sections of 40um, from neural cres)
Permeabilization
Yes - 0.5% triton x-100
Specification
Cryostat tissue sections of 40um, from neural cres
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Paraformaldehyde
Read More

Abcam user community

Verified customer

Submitted Oct 04 2019

Immunohistochemistry (Frozen sections) abreview for Anti-MiTF antibody [C5] - ChIP Grade

 Poor
Abreviews
abreview image
Application
Immunohistochemistry (Frozen sections)
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 37°C
Sample
Mouse Tissue sections (e11.5 embryo transverse cryosection of trunk)
Specification
e11.5 embryo transverse cryosection of trunk
Permeabilization
Yes - 1% saponin
Fixative
Paraformaldehyde
Read More

Abcam user community

Verified customer

Submitted Mar 24 2015

Immunocytochemistry/ Immunofluorescence abreview for Anti-MiTF [C5] antibody - ChIP Grade

 Good
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Blocking step
2.5% goat serum, 2.5% donkey serum, 1% BSA, 0.1% triton as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2.5% · Temperature: 25°C
Sample
Human Cell (neonatal foreskin melanocytes)
Specification
neonatal foreskin melanocytes
Permeabilization
Yes - 0.1% tritonx-100
Fixative
Paraformaldehyde
Read More

Abcam user community

Verified customer

Submitted Apr 16 2014

Question

I'm looking for an MiTF isoform M specific antibody.

Read More
Answer

Thank you for contacting us.
We carry ab12039 and this antibody recognizes serine phosphorylated and non-phosphorylated melanocytic isoforms of microphthalmia. The Mitf antibody ab12039 [C5] recognizes Mouse, Rat, Chicken, Human MiTF in melanocytes. It is a mouse IgG1 Kappa chain antibody. It is believed to be M form specific, but that has not been specifically validated by Abcam. The antibody was originally made from an N terminal peptide coupled to KLH. I would refer you to the references section of our datasheet.
https://www.abcam.com/mitf-antibody-c5-chip-grade-ab12039.html
https://www.abcam.com/MiTF-antibody-C5-ChIP-Grade-ab12039-references.html
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question

I have 2 questions concerning Mitf ab 12039 C5 clone:
- What is the specificity of his antibody, does it recognise only Mitf
isoform M or also Mitf Isoforms A, H, and C?
- In the datasheet is written that the immunogen is N-terminal fragment
of human microphthalmia protein. Is this the N-terminal fragment of
different isoforms of Mitf or of only 1 isoform? And if of only 1
isoform, which one was used?

Read More
Answer

Thank you for your patience.

The following information was obtained:

The Mitf antibody ab12039 [C5] recognizes both human and mouse Mitf in melanocytes. It is a mouse IgG1 Kappa chain antibody. It isbelieved to beM form specific, but wouldneed to be validated that as the literature was to utilize the antibodies C5 and D5 for diagnostic purposes in melanomas. The antibody was originally made from an N terminal peptide coupled to KLH.

I hope this information is of some help to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Question

I have 2 questions concerning Mitf ab 12039 C5 clone:
- What is the specificity of his antibody, does it recognise only Mitf
isoform M or also Mitf Isoforms A, H, and C?
- In the datasheet is written that the immunogen is N-terminal fragment
of human microphthalmia protein. Is this the N-terminal fragment of
different isoforms of Mitf or of only 1 isoform? And if of only 1
isoform, which one was used?

Read More
Answer

Thank you for contacting us.

I have been in touch with the lab. We need to check with the originator of the antibody for these details. I will let you know as soon asI hear back.

Thank you for your patience.

Read More

Immunohistochemistry (Frozen sections) abreview for Anti-MiTF antibody [C5] - ChIP Grade

 Excellent
Abreviews
abreview image
Application
Immunohistochemistry (Frozen sections)
Sample
Chicken Tissue sections (eye)
Specification
eye
Fixative
Paraformaldehyde
Permeabilization
Yes - 1% saponin
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 37°C
Read More

Katia Del Rio-Tsomis

Verified customer

Submitted Sep 30 2011

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-MiTF antibody [C5] - ChIP Grade

 Excellent
Abreviews
abreview image
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (skin)
Specification
skin
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric buffer, pH6
Permeabilization
No
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 20°C
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Abcam user community

Verified customer

Submitted May 26 2011

Question

I performed all the changes you suggested and am still unable to get a band for MITF using the (C5)mouse monoclonal antibody using an even wider panel of melanoma lines and primary human melanocytes. Once again, I am able to get a band for survivin and beta-actin using the same lysates. Could you send me a replacement antibody for MITF from a different lot of the C5 monoclonal?

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Answer

Thank you for your e-mail and informing me you have tried the suggested modifications to your protocol, I appreciate the amount of work you have performed and have immediately arranged a replacement vial to be sent to you. The order is reference 172077 and will be shipped to you today, Could you please let me know how you get on with the replacement vial?

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Question

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 166189 DESCRIPTION OF THE PROBLEM Non-specific bands obtained. Specific bands between 52-70 kD not seen. SAMPLE 3 primary melanocyte lines, and 3 melanoma lines lysed in 2% SDS lysis buffer. PRIMARY ANTIBODY MITF (C5)mouse monoclonal antibody by abcam at 1:1000 dilution in 5%milk for 90 minutes, followed by 3 washes (30 minutes) in PBST. As control, beta actin mouse monoclonal (sigma) and survivin rabbit polyclonal (Dr. Altieri) used similarly. DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED The survivin and beta-actin blots as noted above ANTIBODY STORAGE CONDITIONS antibody aliquoted and stored at -20C. Thawed on ice, used at 1:1000 dilution for western. SAMPLE PREPARATION protease and phosphatase inhibitors (sigma) used. Tris used to pH at 7.4. sample heated at 94C for 3 minutes. AMOUNT OF PROTEIN LOADED 30 micrograms ELECTROPHORESIS/GEL CONDITIONS 10% SDS-PAGE TRANSFER AND BLOCKING CONDITIONS 1X Tris-glycine, 20%methanol used as transfer buffer. Transfer carried out on ice 100V for 1 hour. SECONDARY ANTIBODY [another company] goat anti-mouse (and anti-rabbit for survivin) used at 1:10000 in 5%milk. incubated for 30 minutes, followed by 3 washes in PBST for 30 minutes HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? tried lengthening the incubation and wash steps

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Answer

I'm very sorry to hear you are experiencing problems with ab12039 and thank you for providing your protocol details so we can look into your problem. I would like to recommend the following modifications to your protocol: -use a RIPA or NP40 lysis buffer (the recipe can be found, if you need it, on our protocols page on our website) so as to extract optimally the protein, which is in the nucleus (leaving the samples to lyse for 2-4hours in the lysis buffer containing plenty of fresh protease inhibitors) -block the membrane in 5%BSA in TBST (Tween 20 0.1%) for 1 hour, then rinse lightly the membrane 10 seconds in TBST and incubate the antibody in TBST only. -try diluting the antibody more, you may find that with the above changes you can dilute more. If you still experience problems with the above changes please do not hesitate to contact me and I can arrange for a replacement vial or refund as you purchased the antibody in the last 90 days,

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1-10 of 15 Abreviews or Q&A

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