MITF (Ab-180/73) Antibody

What is MITF and why is it a significant research target?

MITF (Microphthalmia-associated transcription factor) is a critical transcription factor that regulates gene expression essential for cell differentiation, proliferation, and survival. It binds to symmetrical DNA sequences (E-boxes) (5'-CACGTG-3') in the promoters of target genes such as BCL2 and tyrosinase (TYR) . MITF plays particularly important roles in:

• Melanocyte development through regulation of tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1)

• Differentiation of neural crest-derived melanocytes

• Development of mast cells, osteoclasts, and retinal pigment epithelium

• Melanoma research, as indicated by studies validating blockade of MITF function as a potential treatment

What are the key characteristics of the MITF (Ab-180/73) Antibody?

The MITF (Ab-180/73) Antibody is a rabbit polyclonal antibody that specifically recognizes the region around the phosphorylation site of serine 180/73 in the human MITF protein . Key characteristics include:

• Host species: Rabbit

• Clonality: Polyclonal

• Target epitope: Synthesized non-phosphopeptide derived from human MITF around the phosphorylation site of serine 180/73 (P-N-S(p)-P-M)

• Species reactivity: Human and mouse

• Applications: Western blot, immunohistochemistry, immunofluorescence, and ELISA

• Purification method: Affinity-purified from rabbit antiserum using epitope-specific immunogen

• Storage form: Liquid, typically in phosphate buffered saline with sodium azide and glycerol

Which experimental applications is MITF (Ab-180/73) Antibody validated for?

The MITF (Ab-180/73) Antibody has been validated for multiple research applications with specific recommended dilutions :

These validated applications make the antibody versatile for different experimental approaches to study MITF expression and function .

How should I optimize the MITF (Ab-180/73) Antibody for Western blotting experiments?

For optimal Western blot results with MITF (Ab-180/73) Antibody:

• Sample preparation: Begin with 20-30 μg of total protein from your cell/tissue lysate.

• Gel selection: Use 8-10% SDS-PAGE gels, as MITF has a molecular weight of approximately 52-58 kDa .

• Transfer conditions: Transfer to PVDF membrane at 100V for 60-90 minutes in cold transfer buffer.

• Blocking: Block the membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute MITF (Ab-180/73) Antibody at 1:500-1:1000 for initial testing and incubate overnight at 4°C .

• Washing: Wash 3-5 times with TBST, 5 minutes each.

• Secondary antibody: Use HRP-conjugated anti-rabbit IgG at 1:5000-1:10000, incubate for 1 hour at room temperature.

• Detection: Use enhanced chemiluminescence (ECL) for visualization.

• Positive controls: HepG2 cells and COLO205 cells have been validated as expressing detectable levels of MITF .

What are the critical parameters for immunohistochemistry staining with MITF (Ab-180/73) Antibody?

For successful IHC applications with MITF (Ab-180/73) Antibody:

• Tissue preparation: Use formalin-fixed, paraffin-embedded sections (4-6 μm thickness) mounted on positively charged slides.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 15-20 minutes.

• Blocking: Block with 5-10% normal serum from the species of the secondary antibody for 30 minutes.

• Primary antibody: Dilute MITF (Ab-180/73) Antibody at 1:50-1:100 and incubate in a humidified chamber overnight at 4°C .

• Secondary antibody system: Use a biotin-streptavidin-HRP detection system or polymer-based detection for optimal sensitivity.

• Counterstaining: Hematoxylin counterstaining for 30-60 seconds provides good nuclear contrast.

• Positive control: Human skin tissue sections have been validated to show positive MITF staining .

• Visualize with DAB or other appropriate chromogen for 5-10 minutes while monitoring under a microscope to prevent overstaining.

What cell types and models are most appropriate for MITF (Ab-180/73) Antibody research?

Based on the research literature and antibody validation data, the following cell types and models are most appropriate:

• Melanoma cell lines (e.g., SK-MEL, A375, B16) - Express high levels of MITF and are suitable for studying its role in melanoma progression .

• Primary melanocytes - Valuable for studying physiological MITF function in normal melanogenesis.

• HepG2 and COLO205 cells - Validated for Western blot detection of endogenous MITF .

• HeLa cells - Validated for immunofluorescence studies .

• Skin tissue sections - Appropriate for immunohistochemical studies of MITF expression in melanocytes and melanoma tissues .

• In vivo melanoma xenograft models - For studying MITF expression in tumor development and therapeutic responses.

• Transgenic mouse models with MITF mutations - For studying developmental roles of MITF in various tissues.

When selecting appropriate models, consider that MITF expression may vary significantly across different cell types and experimental conditions .

How can I differentiate between MITF isoforms using the MITF (Ab-180/73) Antibody?

MITF exists in multiple isoforms with tissue-specific functions. The MITF (Ab-180/73) Antibody targets a common region around serine 180/73, but additional approaches can help differentiate isoforms:

• Western blot analysis:

  • Use higher percentage gels (10-12%) to achieve better separation of closely migrating isoforms

  • The M isoform (melanocyte-specific) runs at approximately 52-58 kDa

  • Other isoforms (A, B, D, H) have slightly different molecular weights

  • Compare with isoform-specific antibodies when possible

• RT-PCR analysis to complement antibody studies:

  • Design primers specific to different isoform-specific exons

  • Perform quantitative RT-PCR for relative expression of each isoform

  • Validate protein expression patterns observed with the antibody

• siRNA knockdown:

  • Use isoform-specific siRNAs to selectively deplete individual MITF isoforms

  • Confirm specificity of antibody recognition by demonstrating selective loss of specific bands

• Recombinant protein standards:

  • Use recombinant MITF isoforms as standards (available as described in resource )

  • Compare migration patterns with endogenous proteins to identify specific isoforms

The amino acid sequence of MITF Isoform M2 (1-413) provided in resource can be useful for designing isoform-specific detection strategies.

What approaches can resolve discrepancies between FISH and MITF protein expression levels in cancer samples?

Resource describes a situation with HER2 where gene amplification (detected by FISH) doesn't always correlate with protein expression levels. Similar discordance can occur with MITF. To resolve such discrepancies:

• Employ multi-technique validation:

  • Combine FISH for gene amplification with multiple protein detection methods (IHC, Western blot)

  • Use the MITF (Ab-180/73) Antibody at multiple dilutions (1:500-1:3000) to ensure detection sensitivity

  • Consider cell-based ELISA approaches as described in resource

• Quantitative protein analysis:

  • Use quantitative Western blotting with recombinant protein standards

  • Consider digital image analysis of IHC sections to quantify staining intensity

  • Implement the colorimetric cell-based ELISA method described in resource , which provides quantitative data on MITF protein expression

• Analysis of regulatory mechanisms:

  • Investigate post-transcriptional regulation (miRNAs, RNA stability)

  • Examine post-translational modifications affecting protein stability

  • Assess proteasomal degradation rates using inhibitors like MG132

• Statistical analysis:

  • Use multivariate analysis to identify factors contributing to discordance

  • Analyze correlation coefficients between gene copy number and protein levels

  • Establish threshold values that best predict functional outcomes

This multi-faceted approach helps distinguish between clinically significant and insignificant discrepancies between genomic and proteomic data .

How can MITF phosphorylation status be effectively monitored using MITF (Ab-180/73) Antibody?

The MITF (Ab-180/73) Antibody was specifically generated against a region containing the serine 180/73 phosphorylation site, making it potentially useful for monitoring phosphorylation states with some additional techniques:

• Phosphatase treatment controls:

  • Split your samples and treat one set with lambda phosphatase before Western blotting

  • Compare band migration patterns and intensities between treated and untreated samples

  • Shifts in molecular weight can indicate phosphorylation status

• Phospho-specific antibody comparison:

  • Use parallel Western blots with phospho-specific MITF antibodies

  • Compare with total MITF detection using MITF (Ab-180/73) Antibody

  • Calculate phospho-to-total MITF ratios for quantitative analysis

• Phos-tag™ SDS-PAGE:

  • Use Phos-tag™ acrylamide gels to separate phosphorylated from non-phosphorylated proteins

  • Detect using MITF (Ab-180/73) Antibody at 1:500 dilution

  • Multiple bands may indicate different phosphorylation states

• Immunoprecipitation strategy:

  • Immunoprecipitate MITF using MITF (Ab-180/73) Antibody

  • Probe with antibodies against phospho-serine/threonine

  • Alternatively, immunoprecipitate with phospho-specific antibodies and detect with MITF (Ab-180/73)

• Induction of phosphorylation:

  • Treat cells with known inducers of MITF phosphorylation (e.g., UV irradiation, growth factors)

  • Monitor changes in MITF detection pattern using MITF (Ab-180/73) Antibody

  • Compare with parallel phospho-specific detection methods

These approaches can help determine the phosphorylation status of MITF in different experimental conditions and cell types.

What are the most common sources of false positive/negative results with MITF (Ab-180/73) Antibody and how can they be mitigated?

Common issues and solutions include:

• False negatives in Western blotting:

  • Insufficient protein loading - Increase to at least 30 μg total protein

  • Inadequate transfer - Optimize transfer conditions for higher molecular weight proteins

  • Excessive washing - Reduce stringency of washes

  • Degraded antibody - Store antibody according to manufacturer recommendations (-20°C)

  • Solution: Include positive control lysates from HepG2 or COLO205 cells

• False positives in immunohistochemistry:

  • Non-specific binding - Increase blocking time/concentration

  • Endogenous peroxidase activity - Include proper quenching step (3% H₂O₂)

  • Cross-reactivity - Perform peptide competition assays using the immunogen peptide

  • Solution: Include antibody-only negative controls and peptide-blocked controls

• Multiple bands in Western blot:

  • MITF isoforms - Compare with expected molecular weights (52-58 kDa)

  • Degradation products - Add protease inhibitors to lysis buffer

  • Non-specific binding - Optimize blocking and antibody dilution

  • Solution: Validate specific bands with siRNA knockdown experiments

• Weak or inconsistent signals:

  • Antibody dilution too high - Test a dilution series (1:500, 1:1000, 1:2000)

  • Insufficient incubation time - Extend primary antibody incubation (overnight at 4°C)

  • Poor antigen retrieval (for IHC) - Optimize antigen retrieval method

  • Solution: Follow the cell-based ELISA protocol in resource for standardized detection

• Background issues:

  • Insufficient blocking - Increase blocking time to 2 hours

  • Secondary antibody concentration too high - Dilute further

  • Inadequate washing - Increase wash steps to 5x5 minutes with TBST

  • Solution: Use 5% BSA instead of milk for blocking when detecting phosphorylated proteins

How can I establish appropriate experimental controls for MITF research using this antibody?

Robust experimental controls for MITF research should include:

• Positive controls:

  • Cell lines with known MITF expression: HepG2, COLO205, melanoma cell lines

  • Recombinant MITF protein (as described in resource )

  • Tissues with known expression: human skin sections, melanoma samples

• Negative controls:

  • Primary antibody omission control (secondary antibody only)

  • Isotype control (non-specific rabbit IgG at same concentration)

  • Peptide competition/blocking with immunogen peptide

  • MITF-null or MITF-knockdown cells

• Technical validation controls:

  • Loading control detection (GAPDH, β-actin, or tubulin) for Western blots

  • For IHC/IF: internal positive control tissues within sections

  • Serial dilution of lysates to establish linearity of detection

  • Multiple fixation methods to confirm epitope preservation

• Biological validation controls:

  • MITF modulation controls: UV exposure or growth factor treatment to alter MITF levels

  • siRNA/shRNA knockdown of MITF to confirm antibody specificity

  • Cell types with differential MITF expression levels

  • Treatment with MITF inhibitors (as mentioned in resource )

• Specialized controls for phosphorylation studies:

  • Phosphatase-treated samples

  • Kinase inhibitor treatments

  • Stimulation with factors known to induce MITF phosphorylation

Resource describes a cell-based ELISA system that incorporates appropriate positive and negative controls, including GAPDH as an internal control for normalization.

How can I integrate MITF protein expression data with transcriptional activity measurements?

To effectively correlate MITF protein detection with its functional transcriptional activity:

• Parallel protein and mRNA analysis:

  • Detect MITF protein using MITF (Ab-180/73) Antibody by Western blot at 1:500-1:1000 dilution

  • Simultaneously measure MITF mRNA levels by qRT-PCR

  • Compare protein:mRNA ratios across experimental conditions to identify post-transcriptional regulation

• Target gene expression analysis:

  • Measure expression of known MITF target genes (TYR, TYRP1, BCL2)

  • Correlate with MITF protein levels detected by the antibody

  • Analyze using regression analysis to establish quantitative relationships

• Chromatin immunoprecipitation (ChIP) assays:

  • Use MITF (Ab-180/73) Antibody for ChIP to detect MITF binding to target promoters

  • Optimize antibody amount (typically 2-5 μg per ChIP reaction)

  • Correlate ChIP data with target gene expression and MITF protein levels

• Reporter gene assays:

  • Employ luciferase reporters driven by MITF-responsive promoters (e.g., tyrosinase promoter)

  • Correlate reporter activity with MITF protein levels detected by Western blot

  • Establish dose-response relationships between MITF levels and transcriptional output

• Single-cell analysis approaches:

  • Perform immunofluorescence using MITF (Ab-180/73) Antibody at 1:100-1:200 dilution

  • Combine with RNA-FISH for target genes

  • Analyze correlation at single-cell level to account for population heterogeneity

Resource discusses MITF inhibitors that can be used as tools to modulate MITF activity in these integrated analyses. Resource provides information on the E-box DNA sequences that MITF binds to, which is useful for designing ChIP experiments.

How can MITF (Ab-180/73) Antibody be utilized to study MITF's role in melanoma progression and therapy resistance?

To investigate MITF's involvement in melanoma:

• Expression profiling across progression stages:

  • Use Western blot with MITF (Ab-180/73) Antibody to compare MITF levels in:

    • Primary melanocytes

    • Benign nevi

    • Primary melanoma at different stages

    • Metastatic melanoma

  • Correlate expression patterns with clinical outcomes and progression markers

• Therapy response studies:

  • Detect MITF before and after treatment with BRAF/MEK inhibitors

  • Correlate MITF levels with sensitivity/resistance to targeted therapies

  • Use MITF inhibitor compounds as described in resource in combination therapy models

• Functional studies:

  • Manipulate MITF levels (overexpression/knockdown) and monitor changes in:

    • Proliferation

    • Invasion capacity

    • Drug sensitivity

    • Metabolic reprogramming

  • Validate MITF detection using the antibody at 1:500-1:1000 dilution

• Patient-derived xenograft (PDX) models:

  • Analyze MITF expression in PDX models using IHC with MITF (Ab-180/73) Antibody

  • Track expression changes during therapy and relapse

  • Correlate with patient outcomes

• Single-cell analysis:

  • Use immunofluorescence with MITF (Ab-180/73) Antibody at 1:100-1:200

  • Combine with markers of phenotype switching

  • Identify MITF-high and MITF-low subpopulations within tumors

As noted in resource , MITF inhibition has been validated as a potential therapeutic approach for melanoma, making these studies clinically relevant.

What protocols can effectively combine MITF (Ab-180/73) Antibody with other molecular techniques for comprehensive pathway analysis?

For integrated pathway analyses:

• Co-immunoprecipitation studies:

  • Immunoprecipitate MITF using MITF (Ab-180/73) Antibody

  • Optimal antibody amount: 2-5 μg per 500 μg total protein

  • Identify interacting partners by mass spectrometry

  • Validate interactions by reciprocal co-IP and Western blotting

• Proximity ligation assay (PLA):

  • Combine MITF (Ab-180/73) Antibody with antibodies against suspected interacting proteins

  • Use for in situ detection of protein-protein interactions in fixed cells/tissues

  • Optimize antibody dilution to 1:100-1:200 for this application

• ChIP-seq analysis:

  • Use MITF (Ab-180/73) Antibody for chromatin immunoprecipitation

  • Sequence MITF-bound DNA regions

  • Integrate with RNA-seq data to correlate binding with gene expression

  • Analyze for enrichment of E-box motifs (5'-CACGTG-3') as described in resource

• Multiplexed immunofluorescence:

  • Combine MITF detection with markers of:

    • Signaling pathway activation (phospho-ERK, phospho-AKT)

    • Cell cycle regulators

    • Differentiation markers

  • Use spectral unmixing for multi-parameter analysis

  • Dilute MITF (Ab-180/73) Antibody at 1:100-1:200

• Reverse Phase Protein Array (RPPA):

  • Validate MITF (Ab-180/73) Antibody for RPPA applications

  • Integrate with detection of multiple signaling proteins

  • Perform quantitative analysis across sample sets

• CRISPR screens with MITF readout:

  • Use MITF detection by MITF (Ab-180/73) Antibody as phenotypic readout

  • Screen for genes that modulate MITF expression or activity

  • Validate hits using individual knockout/knockdown approaches

Resource provides details on a cell-based ELISA approach that could be adapted for high-throughput screening applications in these integrated analyses.

How can researchers effectively investigate the dynamic regulation of MITF in response to environmental and cellular stressors?

To study MITF's dynamic responses to stressors:

• Time-course analysis protocols:

  • Expose cells to relevant stressors (UV radiation, hypoxia, oxidative stress)

  • Harvest cells at multiple time points (0, 15, 30, 60 min, 2, 4, 8, 24 h)

  • Detect MITF using Western blot with MITF (Ab-180/73) Antibody at 1:500 dilution

  • Quantify changes relative to untreated controls and normalize to appropriate housekeeping proteins

• Live-cell imaging approaches:

  • Create MITF-fluorescent protein fusions (validate with MITF (Ab-180/73) Antibody)

  • Perform time-lapse microscopy during stress exposure

  • Quantify subcellular localization changes and protein dynamics

  • Correlate with functional outcomes

• Post-translational modification analysis:

  • Use phosphatase treatments to detect changes in phosphorylation

  • Employ Phos-tag™ gels to separate differently modified MITF forms

  • Detect with MITF (Ab-180/73) Antibody

  • Correlate modifications with functional changes in MITF activity

• Combination with transcriptional analysis:

  • Perform parallel MITF protein detection and RNA-seq

  • Analyze temporal relationships between MITF protein changes and transcriptional responses

  • Create mathematical models of the temporal dynamics

• Stress-responsive signaling pathway integration:

  • Inhibit or activate specific stress-responsive pathways (p38 MAPK, JNK, etc.)

  • Monitor effects on MITF levels and modifications

  • Use phospho-specific antibodies in parallel with MITF (Ab-180/73) Antibody

  • Construct signaling network models