MITF Antibody, Biotin conjugated

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Description

Key Properties and Reactivity

PropertyDetailsSources
ReactivityHuman, canine, dog; negative for mouse and rat in some formulations .
ConjugateBiotin for enhanced detection sensitivity
LocalizationNuclear, as MITF functions as a transcription factor
Isoforms RecognizedMITF-M (melanocyte-specific), MITF-A, -B, -C, -H

Applications in Research

The antibody is validated for diverse techniques:

ApplicationKey DetailsSources
ImmunohistochemistryDetects MITF in melanomas, nevi, and retinal pigment epithelium. Optimal dilutions vary (e.g., 1:200–400 for IHC) .
Flow CytometryUsed to analyze MITF expression in melanocytic cells. Requires empirical optimization.
ChIP-SeqAbFlex® MITF antibody (Active Motif) validates MITF-DNA binding in epigenetic studies.
Western BlottingPolyclonal antibodies (e.g., ABIN676373) detect phosphorylated MITF isoforms.

Research Findings and Mechanistic Insights

  • Role in Melanoma: MITF regulates melanocyte survival and pigmentation. Its upregulation is linked to melanoma progression, and antibodies help study its interaction with oncogenic pathways (e.g., BRAF V600E) .

  • Transcriptional Regulation: ATF2 negatively regulates MITF by suppressing SOX10 transcription, impacting melanoma development .

  • Post-Translational Modifications: Phosphorylation by MAP kinase enhances MITF’s transcriptional activity, which can be studied using phosphorylation-specific antibodies .

Product Specs

Buffer
Preservative: 0.03% Proclin 300
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Form
Liquid
Lead Time
Typically, we can ship your order within 1-3 business days after receiving it. However, delivery times may vary depending on the method of purchase and location. For specific delivery times, we recommend consulting your local distributors.
Synonyms
Microphthalmia-associated transcription factor (Class E basic helix-loop-helix protein 32) (bHLHe32), MITF, BHLHE32
Target Names
Uniprot No.

Target Background

Function
MITF is a transcription factor that plays a crucial role in regulating the expression of genes essential for cell differentiation, proliferation, and survival. It binds to M-boxes (5'-TCATGTG-3') and symmetrical DNA sequences (E-boxes) (5'-CACGTG-3') located in the promoters of target genes, including BCL2 and tyrosinase (TYR). MITF is a key player in melanocyte development, regulating the expression of tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1). Additionally, it plays a critical role in the differentiation of various cell types, such as neural crest-derived melanocytes, mast cells, osteoclasts, and optic cup-derived retinal pigment epithelium.
Gene References Into Functions
  1. Research indicates that MITF is highly expressed in myeloma cells and regulates cdk2 expression, contributing to cell resistance to both BRAF and Hsp90 inhibitors. PMID: 29507054
  2. Evidence suggests that glycogen synthase kinase 3 (GSK3) and proto-oncogene proteins B-raf (BRAF)/MAPK signaling converge to control microphthalmia-associated transcription factor MITF (MITF) nuclear export. PMID: 30150413
  3. Our findings provide new insights into how MITF mutations can lead to different phenotypes of WS2 through the Wnt/beta-catenin signaling pathway. PMID: 29531335
  4. This study reports a novel mutation, c.718C>G; p. (Arg240Gly) in the melanogenesis associated transcription factor gene, in Han people with hearing loss. PMID: 29484430
  5. The study examined the upregulation of microphthalmia-associated transcription factor (Mitf) and enhanced melanogenesis by Cymbopogon schoenanthus phenol extracts. PMID: 29359158
  6. The essential melanocyte-specific transcription factor MITF regulates the expression of the MYO5A gene, which encodes the molecular motor myosin-Va. PMID: 27939378
  7. These findings suggest that primary and metastatic melanomas contain not only MITF-high and MITF-low cells but also subpopulations expressing markers of both signatures. The coexistence of these three cell populations, whether adjacent or intermixed, contributes to the spatial heterogeneity of the tumors. PMID: 28855355
  8. The SH3BP4 gene is transcriptionally regulated by MITF as its direct target. PMID: 28819321
  9. This research demonstrates that the FANC pathway operates downstream of MiTF, establishing an epistatic relationship between MiTF and the FANC pathway. PMID: 27827420
  10. MITF expression levels in hepatic cancer cells may be determined by the balance between Hedgehog signaling and cellular stress. PMID: 28794318
  11. Data strongly suggest that glucose deprivation suppresses MITF expression through reactive oxygen species-induced ATF4 up-regulation, resulting in reduced melanoma cell proliferation. PMID: 28380427
  12. The study showed that overexpression of MITF-A leads to a significant increase in nephron number and larger kidneys, while Mitfa deficiency results in a reduced nephron number. PMID: 29240767
  13. MITF may play a role in the development of acquired drug resistance through hyper-activation of the PI3K pathway. PMID: 27391157
  14. Mutations in the MITF gene are associated with Waardenburg syndrome type 2A. PMID: 29094203
  15. A sumoylation-defective germline mutation in microphthalmia-associated transcription factor (MITF), a master regulator of melanocyte homeostasis, is associated with the development of melanoma. [review] PMID: 28825724
  16. Single Nucleotide Polymorphism in the MITF gene is associated with facial solar lentigines. PMID: 27327535
  17. Phosphorylation of MITF by AKT affects its downstream targets and causes TP53-dependent cell senescence. PMID: 27702651
  18. This research identified two novel MITF mutations in patients with TS/WS2A. The findings suggest that posterior microphthalmos might be part of the clinical characteristics of Tietz/Waardenburg syndrome type 2A and expand both the clinical and molecular spectrum of the disease. PMID: 27604145
  19. Data show that poly(ADP-ribose) polymerase 1 (PARP1)-mediated senescence rescue was accompanied by transcriptional activation of the melanocyte-lineage survival oncogene MITF, indicating a role for PARP1 in melanomagenesis. PMID: 28759004
  20. MITF is a direct target of miR-137. PMID: 26845432
  21. The study found in melanoma cell lines that ILEI is highly expressed in MITF-low invasive cells, and that phenotype switching between the MITF-low invasive state and the MITF-high proliferative state can alter ILEI expression. PMID: 28545079
  22. Suppression of MITF activity by UCHL1 via protein degradation might contribute to the development of new therapeutic approaches for melanoma or dyspigmentation disorders. PMID: 28392346
  23. The results of this study have provided new and surprising insights into the effect of Bcl-2 overexpression in melanoma cells, namely that Bcl-2 modulates MITF nuclear activity. PMID: 26599548
  24. This research provides insights into molecular interactions between CRD-BP and MITF mRNA. PMID: 28182633
  25. These data uncover novel mechanisms linking MITF-dependent inhibition of invasion to suppression of guanylate metabolism. PMID: 27181209
  26. This research demonstrates that AR can promote melanoma metastasis by altering the miRNA-539-3p/USP13/MITF/AXL signal, and targeting this newly identified signal with AR degradation enhancer ASC-J9 may help to better suppress melanoma metastasis. PMID: 27869170
  27. This review examines the fundamental functions of MITF in melanocytes and melanoma. PMID: 28263292
  28. Our results demonstrate that MITF-E318K reduces the program of senescence, potentially favoring melanoma progression in vivo. PMID: 28376192
  29. The germline variant MITF, p.E318K is associated with an increased risk of other neural crest-derived tumors, such as PCC/PGL. PMID: 27680874
  30. Microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B. PMID: 28096186
  31. GPER enhances melanogenesis via PKA by upregulating microphthalmia-related transcription factor-tyrosinase in melanoma. PMID: 27378491
  32. Data indicate that TFAP2A binds to many of the same regulatory elements as MITF in melanocytes. PMID: 28249010
  33. This study describes a syndrome, termed COMMAD, characterized by coloboma, osteopetrosis, microphthalmia, macrocephaly, albinism, and deafness; COMMAD is associated with biallelic MITF mutant alleles, suggesting a role for MITF in regulating processes such as optic-fissure closure and bone development or homeostasis, which extend beyond what is typically observed in individuals with monoallelic MITF mutations. PMID: 27889061
  34. Data suggest that NFIB protein increases EZH2 protein expression downstream of BRN2 protein, which further decreases MITF protein levels. PMID: 28119061
  35. In melanoma lymph node metastases, MITF protein expression was not tightly correlated with its gene targets. PMID: 27515936
  36. DRD4 antagonist exhibits an antimelanogenic effect related to downregulation of MITF transcription through activation of the ERK pathway. PMID: 26782007
  37. Akt modulates nuclear translocation of MITF. PMID: 28165011
  38. This research has established that the cooperative antiproliferative effects of aspirin and I3C in human melanoma cells trigger a significant downregulation of MITF-M gene expression and disruption of MITF-M promoter activity. The findings demonstrate that aspirin-regulated Wnt signaling and I3C-targeted signaling pathways converge at distinct DNA elements in the MITF-M promoter to cooperatively disrupt MITF-M expression. PMID: 27055402
  39. The addition of MITF>/=50% into the logistic regression analysis significantly improves the accuracy of the melanoma nomogram in predicting regional nodal spread. PMID: 27919990
  40. The MITF p.E318K mutation does not appear to play a major role in sporadic renal cell carcinoma carcinogenesis, but is possibly restricted to a rare subpopulation of inherited renal cell carcinoma. PMID: 26999813
  41. Overexpression of MITF is associated with melanoma cell survival and progression. PMID: 27185926
  42. This study concludes that the expression of Rlbp1 and Rdh5 critically depends on functional Mitf in the RPE and suggests that MITF plays a significant role in controlling retinoid processing in the RPE. PMID: 26876013
  43. This study demonstrated that concomitant AURKA/BRAF and AURKA/MEK targeting overcame MAPK signaling activation-associated resistance signature in BRAF- and NRAS-mutated melanomas, respectively, leading to heightened anti-proliferative activity and apoptotic cell death. PMID: 26962685
  44. This study found that Mitf, likely including Mitf-M, is expressed in the mitral cells and tufted cells that transmit information from olfactory sensory neurons to the olfactory cortex. PMID: 26522736
  45. SOX5 has a strong inhibitory effect on MITF expression and appears to have a decisive clinical impact on melanoma during tumor progression. PMID: 26927636
  46. In addition to melanoma risk, MITF p.E318K is associated with a high nevi count and could play a role in fast-growing melanomas. PMID: 26650189
  47. The expression of the molecular marker Mitf in primary cutaneous melanomas is a useful tool for assessing lymph node status. PMID: 26317170
  48. This study proposes an MITF-CEACAM1 axis as a potential determinant of melanoma progression. PMID: 26301891
  49. LEF-1 and MITF regulate tyrosinase gene transcription in vitro by binding to its promoter. PMID: 26580798
  50. This study shows that MITF-A mRNA is predominantly expressed in all three human liver cancer cell lines examined. PMID: 26773496

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Database Links

HGNC: 7105

OMIM: 103470

KEGG: hsa:4286

STRING: 9606.ENSP00000295600

UniGene: Hs.166017

Involvement In Disease
Waardenburg syndrome 2A (WS2A); Waardenburg syndrome 2, with ocular albinism, autosomal recessive (WS2-OA); Tietz albinism-deafness syndrome (TADS); Melanoma, cutaneous malignant 8 (CMM8); Coloboma, osteopetrosis, microphthalmia, macrocephaly, albinism, and deafness (COMMAD)
Protein Families
MiT/TFE family
Subcellular Location
Nucleus. Cytoplasm.
Tissue Specificity
Expressed in melanocytes (at protein level).; [Isoform A2]: Expressed in the retinal pigment epithelium, brain, and placenta. Expressed in the kidney.; [Isoform C2]: Expressed in the kidney and retinal pigment epithelium.; [Isoform H1]: Expressed in the k

Q&A

What is MITF and why are biotin-conjugated antibodies used in its detection?

MITF (Microphthalmia-associated Transcription Factor) is a basic helix-loop-helix-leucine-zipper (bHLH-Zip) transcription factor that regulates the development and survival of melanocytes and retinal pigment epithelium. It plays critical roles in transcription of pigmentation enzyme genes such as tyrosinase, TRP1, and TRP2 . Biotin-conjugated antibodies are used because biotin provides signal amplification through its high-affinity interaction with streptavidin, enhancing detection sensitivity in techniques like IHC, flow cytometry, and immunofluorescence without requiring direct enzyme conjugation to the primary antibody. The biotin-streptavidin system allows for flexible detection protocols and improved signal-to-noise ratios in experiments targeting nuclear proteins like MITF.

How do different MITF isoforms impact antibody selection?

Multiple isoforms of MITF exist, including MITF-A, MITF-B, MITF-C, MITF-H, and MITF-M, which differ in their amino-terminal domains and expression patterns . When selecting a biotin-conjugated MITF antibody, researchers should consider which isoform is relevant to their study. For instance:

  • MITF-M isoform is restricted to the melanocyte cell lineage

  • Other isoforms have broader expression patterns

This isoform diversity necessitates careful review of the antibody immunogen sequence. For example, antibodies targeting amino acids 1-114 may recognize different isoforms than those targeting amino acids 351-450 . Consult the manufacturer's specificity data to ensure the antibody recognizes your isoform of interest.

What are the optimal protocols for using biotin-conjugated MITF antibodies in immunohistochemistry?

For optimal immunohistochemistry results with biotin-conjugated MITF antibodies:

  • Antigen retrieval: Heat-mediated antigen retrieval in citrate buffer (pH 6.0) is typically recommended for formalin-fixed, paraffin-embedded (FFPE) tissues.

  • Blocking: Implement two blocking steps:

    • Block endogenous biotin using a commercial avidin/biotin blocking kit

    • Block non-specific binding with 1-5% BSA or serum from the species of the secondary detection reagent

  • Antibody dilution: Experimentally determine optimal dilution, typically starting with 1:200-400 for polyclonal or according to manufacturer recommendations for monoclonal antibodies.

  • Detection system: Use streptavidin-HRP or streptavidin-fluorophore conjugates depending on desired visualization method.

  • Nuclear counterstain: Use DAPI or hematoxylin to contrast the nuclear localization of MITF.

Remember that MITF is localized to the nucleus, so proper nuclear visualization is critical for accurate interpretation .

How should biotin-conjugated MITF antibodies be optimized for flow cytometry applications?

For flow cytometry applications with biotin-conjugated MITF antibodies:

  • Cell preparation: Since MITF is a nuclear protein, ensure proper permeabilization of cells with 0.1% Triton X-100 or commercial permeabilization buffers.

  • Fixation protocol: Fix cells with 2-4% paraformaldehyde for 10-15 minutes at room temperature.

  • Antibody concentration: Titrate the antibody to determine optimal concentration. Start with the manufacturer's recommendation and test 2-fold dilutions.

  • Detection strategy: Use streptavidin conjugated to fluorophores with appropriate excitation/emission spectra compatible with your cytometer configuration.

  • Controls: Include:

    • Isotype controls conjugated to biotin

    • Cells known to be positive (melanoma cell lines) and negative (non-melanocytic cells) for MITF expression

    • Blocking peptide controls to verify specificity

For clones like MITF/2987R and D5, preliminary testing should be conducted to determine optimal antibody concentration for your specific cell type .

How can researchers address non-specific binding issues with biotin-conjugated MITF antibodies?

Non-specific binding is a common challenge with biotin-conjugated antibodies due to endogenous biotin in tissues. To address this:

  • Endogenous biotin blocking: Use a commercial avidin/biotin blocking kit before antibody incubation.

  • Tissue-specific considerations: Tissues with high endogenous biotin (liver, kidney, brain) require more rigorous blocking protocols.

  • Antibody validation: Confirm specificity using:

    • Western blotting with expected band at ~52-54 kDa

    • Peptide competition assays

    • siRNA knockdown validation

  • Protocol optimization: If background persists:

    • Increase blocking time and concentration (3-5% BSA)

    • Reduce primary antibody concentration

    • Include 0.1-0.3% Triton X-100 in antibody diluent

    • Extend washing steps (5x5 minutes)

  • Alternative detection: Consider tyramide signal amplification systems that provide high sensitivity with reduced background.

These approaches can help distinguish true MITF nuclear staining from artifacts.

What controls should be included when performing experiments with biotin-conjugated MITF antibodies?

Rigorous experimental controls are essential:

Control TypePurposeImplementation
Positive controlVerify antibody functionalityUse melanoma cell lines or tissues known to express MITF
Negative controlAssess non-specific bindingUse tissues/cells known to be MITF-negative (e.g., mouse and rat samples for clone MITF/2987R)
Isotype controlEvaluate background from antibody classInclude species-matched irrelevant antibody with same conjugate
Secondary-only controlDetermine background from detection systemOmit primary antibody
Blocking peptide controlConfirm epitope specificityPre-incubate antibody with immunizing peptide
siRNA/knockout validationVerify target specificityCompare staining in MITF-knockdown vs. wild-type cells

For MITF antibodies specifically, nuclear localization pattern should be evident in positive cells. Any cytoplasmic staining should be carefully scrutinized as potential non-specific binding .

How can biotin-conjugated MITF antibodies be utilized in investigating DNA damage response in melanoma?

Recent research has revealed MITF's non-transcriptional role in DNA damage response (DDR) in melanoma . Biotin-conjugated MITF antibodies can be instrumental in studying this phenomenon:

  • Co-localization studies: Use biotin-conjugated MITF antibodies with streptavidin-fluorophores in multi-color immunofluorescence to visualize MITF co-localization with DDR proteins (γH2AX, 53BP1, RAD51) at sites of DNA damage.

  • Chromatin immunoprecipitation (ChIP): Employ biotin-conjugated MITF antibodies to assess MITF binding to chromatin at damage sites, coupled with streptavidin-magnetic beads for pulldown.

  • Proximity ligation assays (PLA): Investigate protein-protein interactions between MITF and MRN complex components after DNA damage using biotin-conjugated MITF antibodies with appropriate PLA probes.

  • Flow cytometry analysis: Quantify changes in MITF levels in response to DNA-damaging agents across cell populations.

These approaches can help elucidate how MITF phosphorylation by ATM/DNA-PK affects its function and localization at sites of DNA damage, potentially contributing to genome instability in melanoma .

What are the considerations for multiplex immunofluorescence when using biotin-conjugated MITF antibodies?

Multiplex immunofluorescence with biotin-conjugated MITF antibodies requires careful planning:

  • Sequential labeling strategy: Since biotin amplification systems can cause cross-reactivity in multiplex settings, consider:

    • Sequential detection with complete stripping between rounds

    • Tyramide signal amplification systems that allow heat-mediated deactivation

    • Spectral unmixing to resolve overlapping signals

  • Panel design considerations:

    • Include markers for cell type identification (melanocyte/melanoma markers)

    • Add DDR pathway components if studying DNA damage (γH2AX, 53BP1)

    • Consider proliferation markers (Ki-67) to correlate with MITF expression

  • Optimization parameters:

    • Antibody order (typically from weakest to strongest signal)

    • Biotin blocking between rounds if using multiple biotinylated antibodies

    • Fixation between detection steps to prevent antibody dissociation

  • Data analysis approach:

    • Use single-stained controls for spectral unmixing

    • Implement cell segmentation algorithms that can distinguish nuclear vs. cytoplasmic signals

    • Quantify co-localization coefficients when studying protein-protein interactions

These strategies can enable complex studies of MITF in relation to other proteins while maintaining specificity and sensitivity.

How do monoclonal vs. polyclonal biotin-conjugated MITF antibodies compare in research applications?

Both monoclonal and polyclonal biotin-conjugated MITF antibodies have distinct advantages:

CharacteristicMonoclonal (e.g., D5, MITF/2987R)Polyclonal (e.g., bs-1990R-Biotin)
SpecificityHigher target specificity; recognizes single epitopeRecognizes multiple epitopes; potentially higher sensitivity
Batch consistencyHigh lot-to-lot reproducibilityGreater batch variation
Species reactivityOften limited (e.g., MITF/2987R: human and canine; negative for mouse/rat) Often broader (e.g., bs-1990R: mouse with predicted reactivity to human, rat, dog, etc.)
ApplicationsExcellent for quantitative studies requiring consistent resultsBetter for detection of proteins in denatured/fixed samples
Target isoformsMay be isoform-specific depending on epitope locationTypically recognizes multiple isoforms
Signal-to-noiseOften cleaner backgroundMay have higher background

Selection should be based on experimental goals: monoclonals for precise quantitation and reproducibility; polyclonals when maximizing detection sensitivity is paramount or when protein conformation may be altered by experimental conditions.

What technical considerations exist when investigating MITF phosphorylation states using biotin-conjugated antibodies?

Investigating MITF phosphorylation states requires specific technical approaches:

  • Phosphorylation-specific antibodies: While standard MITF antibodies detect total protein, phospho-specific antibodies (pSer73, pSer180) should be used alongside biotin-conjugated general MITF antibodies to determine phosphorylation status .

  • Sample preparation considerations:

    • Include phosphatase inhibitors in all buffers

    • Minimize time between sample collection and fixation/lysis

    • Consider lambda phosphatase treatment as negative control

  • Activation conditions: MITF phosphorylation increases after:

    • Exposure to DNA-damaging agents (activates ATM/DNA-PK pathways)

    • c-Kit activation (activates MAP kinase pathway)

  • Functional significance: Phosphorylated MITF:

    • Dissociates from transcription cofactors

    • Stabilizes at DNA damage sites

    • Interacts with MRN complex

    • Limits HR-mediated repair

  • Comparison with MITF-E318K: The melanoma-predisposing MITF-E318K mutation recapitulates effects of phosphorylated MITF, suggesting a mechanistic link to increased melanoma risk .

These considerations are essential for accurately interpreting MITF's phosphorylation-dependent roles in transcriptional regulation and DNA damage response.

How might biotin-conjugated MITF antibodies be utilized in emerging single-cell technologies?

Biotin-conjugated MITF antibodies have potential applications in emerging single-cell technologies:

  • Single-cell proteomics: Integration with mass cytometry (CyTOF) using metal-tagged streptavidin to quantify MITF levels alongside dozens of other proteins at single-cell resolution.

  • Spatial transcriptomics: Combination with in situ transcriptomics to correlate MITF protein localization with gene expression patterns in tissue sections.

  • Microfluidic applications: Implementation in droplet-based single-cell protein analysis platforms using streptavidin-based detection systems.

  • Live-cell imaging: Development of cell-permeable biotin-conjugated antibody fragments to track MITF dynamics in living cells.

  • Clonal analysis: Use in colony assays to identify clonal populations with variable MITF expression and correlate with functional phenotypes.

These approaches could provide unprecedented insights into MITF's role in cellular heterogeneity within melanocytic lesions and melanomas, particularly regarding the relationship between MITF expression levels and DNA damage accumulation .

What are the methodological considerations for using biotin-conjugated MITF antibodies in chromatin immunoprecipitation studies?

For chromatin immunoprecipitation (ChIP) applications with biotin-conjugated MITF antibodies:

  • Crosslinking optimization: Standard formaldehyde crosslinking (1%) for 10 minutes typically works for transcription factors like MITF, but optimization may be needed.

  • Sonication parameters: Target chromatin fragments of 200-500bp, verifying by gel electrophoresis before immunoprecipitation.

  • Pre-clearing strategy: Include a pre-clearing step with protein A/G beads to reduce non-specific binding.

  • Pulldown approach: Two viable methods:

    • Direct method: Use streptavidin-coated magnetic beads to capture biotin-conjugated MITF antibodies

    • Indirect method: Use protein A/G beads with a secondary antibody that recognizes the MITF antibody

  • Control selection:

    • Input control (pre-immunoprecipitation chromatin)

    • IgG-biotin control (non-specific pulldown)

    • Positive control (known MITF binding sites, e.g., TYR, TYRP1 promoters)

  • Analysis considerations:

    • qPCR for known targets

    • ChIP-seq for genome-wide binding profiles

    • Integrate with DNA damage markers when studying MITF's role in DNA repair

These methodological considerations are critical for successfully using biotin-conjugated MITF antibodies in chromatin studies that investigate both transcriptional and non-transcriptional functions.

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