MAFK Antibody

What is MAFK and what is its molecular function?

MAFK (Transcription factor MafK) is a member of the Maf family of basic-leucine zipper (bZIP) transcription factors, specifically belonging to the small Maf subfamily along with MafF and MafG. Small Maf proteins like MAFK lack the N-terminal acidic domain important for transcriptional activation that is present in large Maf family members (such as c-Maf, Nrl, MafA, and MafB). MAFK has a molecular weight of approximately 18 kDa (17,523 Da) .

What types of MAFK antibodies are currently available for research applications?

Research-grade MAFK antibodies are available in several formats including:

• Monoclonal antibodies: These include mouse monoclonal antibodies such as clone 381923 (R&D Systems) and clone 1328CT786.105.125 (Boster Bio)

• ELISA kits: For quantitative detection of MAFK in various sample types, including competitive ELISA formats with detection ranges of 0.5-10 ng/mL and sensitivity of 0.1 ng/mL

These antibodies have been validated for various applications including Western blot, flow cytometry, and ELISA across human and mouse samples .

What are the common alternative names for MAFK that researchers should be aware of?

When searching literature and reagents, researchers should be aware that MAFK is also known by several alternative names:

• NFE2U

• P18

• Erythroid transcription factor NF-E2 p18 subunit

• Nuclear factor erythroid-2, ubiquitous (p18)

• Basic-leucine zipper transcription factor MafK

• v-maf avian musculoaponeurotic fibrosarcoma oncogene family, protein K

• v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog K

• v-maf musculoaponeurotic fibrosarcoma oncogene homolog K

Understanding these alternative designations is crucial for comprehensive literature searches and proper experimental design.

What are the validated applications for MAFK antibodies in research?

Current MAFK antibodies have been validated for several applications:

When designing experiments, researchers should select antibodies validated for their specific application and sample type to ensure reliable results .

What is the optimal protocol for Western blot detection of MAFK?

For optimal Western blot detection of MAFK, researchers should consider the following validated protocol:

• Sample preparation: Prepare cell or tissue lysates under reducing conditions

• Gel electrophoresis: Load 35 μg of lysate per lane on SDS-PAGE

• Transfer: Use PVDF membrane for optimal protein binding

• Blocking: Follow antibody manufacturer's recommendation (typically 5% non-fat milk or BSA)

• Primary antibody: Dilute anti-MAFK antibody to appropriate concentration (1:1000 for Boster Bio M06288; 2 μg/mL for R&D Systems MAB3809)

• Secondary antibody: Use appropriate HRP-conjugated secondary antibody (e.g., goat anti-mouse IgG HRP at 1:3000)

• Detection: Use enhanced chemiluminescence (ECL) for visualization

• Expected band: Look for specific band at approximately 18 kDa

The specificity of anti-MAFK antibodies should be verified using positive controls like Jurkat or A549 cell lysates, where the antibody has been demonstrated to detect MAFK successfully .

How should researchers optimize flow cytometry analysis using MAFK antibodies?

For flow cytometry applications with MAFK antibodies, consider this optimized approach:

• Cell preparation: Harvest cells (e.g., HeLa) and fix/permeabilize according to standard protocols for intracellular staining of nuclear proteins

• Blocking: Block with appropriate buffer to prevent non-specific binding

• Primary antibody: Incubate with anti-MAFK antibody (e.g., Boster Bio M06288) at 1:25 dilution

• Secondary antibody: Use fluorochrome-conjugated secondary antibody such as Alexa Fluor 488 goat anti-mouse IgG at 1:400 dilution

• Washing: Perform thorough washing steps between incubations

• Controls: Always include isotype control (e.g., mouse IgG1) and unstained samples

• Analysis: Analyze using standard flow cytometry instrumentation and software

This protocol has been validated with HeLa cells and has demonstrated specific detection of MAFK compared to isotype controls .

How can researchers validate the specificity of MAFK antibodies?

Validating antibody specificity is crucial for reliable research results. For MAFK antibodies, consider these validation approaches:

• Positive control samples: Use cell lines with known MAFK expression (e.g., Jurkat, A549, HeLa)

• Recombinant protein controls: Include recombinant MAFK protein as a positive control

• Cross-reactivity testing: Test against related proteins (MafB, MafF, MafG) to ensure specificity

• Western blot analysis: Confirm detection of a single band at the expected molecular weight (~18 kDa)

• Isotype controls: For flow cytometry applications, include appropriate isotype controls (e.g., mouse IgG1)

• Knockout/knockdown validation: If possible, use MAFK knockout or knockdown samples as negative controls

R&D Systems has demonstrated the specificity of their MAFK antibody (MAB3809) by showing it detects recombinant MAFK but not related family members such as MafB, MafF, and MafG in Western blot analysis .

What are common issues with MAFK detection and how can they be resolved?

Several challenges can arise when working with MAFK antibodies:

When troubleshooting, always include positive controls that have been previously validated with the specific antibody being used .

What is the recommended approach for antibody storage and handling to maintain activity?

Proper storage and handling of MAFK antibodies is essential for maintaining their activity and specificity:

• Storage temperature:

  • Long-term: -20°C to -70°C (for up to 12 months from receipt)

  • Medium-term (reconstituted antibodies): -20°C to -70°C for up to 6 months

  • Short-term: 2-8°C for up to 1 month after reconstitution

• Handling recommendations:

  • Avoid repeated freeze-thaw cycles

  • Use a manual defrost freezer

  • Store in small aliquots after reconstitution

  • Keep antibodies under sterile conditions after reconstitution

  • Follow manufacturer's specific instructions for each antibody format

Following these guidelines will help ensure consistent antibody performance across experiments.

How can MAFK antibodies be utilized in studying MAFK's role in protein-protein interactions?

MAFK functions primarily through interactions with other transcription factors. Researchers can leverage MAFK antibodies to study these interactions through:

• Co-immunoprecipitation (Co-IP): Use anti-MAFK antibodies to pull down MAFK and its binding partners, followed by Western blot or mass spectrometry analysis to identify interacting proteins.

• Chromatin immunoprecipitation (ChIP): Employ anti-MAFK antibodies to identify genomic binding sites of MAFK and its dimerization partners, particularly when studying heterodimers with p45 NF-E2 that promote globin gene expression.

• Proximity ligation assay (PLA): Combine MAFK antibodies with antibodies against suspected interaction partners to visualize and quantify protein-protein interactions in situ.

• Bimolecular fluorescence complementation (BiFC): Tag potential interaction partners with complementary fluorescent protein fragments and use MAFK antibodies to validate expression and localization.

These approaches can provide insights into how MAFK's transcriptional repression or activation functions are regulated through protein-protein interactions with other bZIP factors .

What are the considerations for using MAFK antibodies in multi-parameter flow cytometry?

When incorporating MAFK antibodies into multi-parameter flow cytometry panels:

• Panel design: Consider fluorochrome selection carefully to minimize spectral overlap with other markers in your panel. Since MAFK is typically detected with secondary antibodies, choose a fluorochrome that complements your existing panel.

• Compensation: Perform proper compensation using single-stained controls, especially important when combining MAFK detection with multiple other markers.

• Fixation and permeabilization: Optimize protocols to ensure access to nuclear MAFK while preserving other cellular epitopes of interest.

• Titration: Determine the optimal concentration of MAFK antibody (starting at 1:25 dilution) for your specific experimental conditions to maximize signal-to-noise ratio.

• Gating strategy: Develop a comprehensive gating strategy that accounts for potential non-specific binding, using appropriate isotype controls (mouse IgG1 for the Boster Bio antibody).

Researchers have successfully applied these considerations when analyzing MAFK expression in HeLa cells using flow cytometry with the Boster Bio M06288 antibody at 1:25 dilution and an Alexa Fluor 488 secondary antibody .

How can researchers integrate MAFK antibody-based detection with genomic approaches?

Integrating protein-level detection of MAFK with genomic approaches can provide comprehensive insights into MAFK function:

• ChIP-seq: Use MAFK antibodies for chromatin immunoprecipitation followed by next-generation sequencing to map genome-wide binding sites of MAFK, particularly in the context of its role in transcriptional regulation.

• CUT&RUN or CUT&Tag: Apply MAFK antibodies in these newer techniques, which offer improved signal-to-noise ratios compared to traditional ChIP for mapping DNA binding sites.

• RNA-seq with protein validation: Combine transcriptome analysis via RNA-seq with MAFK protein detection via Western blot or immunofluorescence to correlate MAFK expression levels with transcriptional changes.

• Proteogenomic integration: Correlate MAFK binding sites (from ChIP-seq) with changes in target gene expression (from RNA-seq) and validate with MAFK protein quantification (via ELISA or Western blot).

This integrated approach allows researchers to connect MAFK's physical presence at genomic loci with functional outcomes in gene expression, providing deeper insights into its role as a transcription factor that can act as either a repressor or activator depending on its dimerization partners .

What controls should be included when working with MAFK antibodies?

Proper experimental controls are essential for generating reliable and interpretable results with MAFK antibodies:

How should researchers select between different MAFK antibody clones for specific applications?

Selection of the appropriate MAFK antibody clone depends on several factors:

• Target epitope: Different clones target different regions of MAFK. The Boster Bio antibody (M06288) is generated against a synthetic peptide from the human region of MAFK, while the R&D Systems antibody (MAB3809) uses recombinant human MAFK (Thr2-Ser156) as immunogen .

• Validated applications: Choose antibodies specifically validated for your application of interest:

  • For Western blot: Both Boster Bio M06288 and R&D Systems MAB3809 are validated

  • For flow cytometry: Boster Bio M06288 is validated

  • For ELISA: Consider specialized ELISA kits

• Species reactivity: Ensure compatibility with your experimental model:

  • The Boster Bio antibody is reactive to human MAFK

  • The R&D Systems antibody recognizes both human and mouse MAFK

• Published validation: Consider antibodies with peer-reviewed publications supporting their use, such as the R&D Systems MAB3809 antibody cited in Bone research .

Researchers should review the technical documentation for each antibody clone and, when possible, test multiple clones in preliminary experiments to determine which performs best in their specific experimental system.

What are the key considerations for quantitative analysis of MAFK expression levels?

For accurate quantitative analysis of MAFK expression:

• Sample preparation consistency:

  • For Western blot: Standardize lysis conditions and protein extraction protocols

  • For flow cytometry: Maintain consistent fixation and permeabilization procedures

  • For ELISA: Follow manufacturer recommendations for sample dilution (neat, 1:2, or 1:4 for the MBS7234931 kit)

• Standard curves and calibration:

  • Use recombinant MAFK protein at known concentrations to generate standard curves

  • For ELISA: The competitive ELISA format (MBS7234931) offers a detection range of 0.5-10 ng/mL and sensitivity of 0.1 ng/mL

• Normalization strategies:

  • For Western blot: Normalize to housekeeping proteins or total protein

  • For flow cytometry: Use appropriate reference populations and account for autofluorescence

  • For cell-based assays: Normalize to cell number or total protein content

• Quantification methods:

  • For Western blot: Use densitometry with appropriate software, including linear range validation

  • For flow cytometry: Report median fluorescence intensity (MFI) rather than percent positive

  • For ELISA: Ensure samples fall within the linear range of the standard curve

• Statistical analysis:

  • Perform at least three independent biological replicates

  • Apply appropriate statistical tests based on data distribution

  • Consider power analysis to determine adequate sample size

Following these guidelines will improve the accuracy and reproducibility of quantitative MAFK expression analyses.

How can MAFK antibodies be applied in studies of erythroid differentiation and hematopoiesis?

MAFK plays a significant role in erythroid differentiation through its heterodimerization with p45 NF-E2. Researchers investigating hematopoiesis can apply MAFK antibodies in several ways:

• Tracking MAFK expression during erythroid differentiation:

  • Use Western blot or flow cytometry to monitor MAFK levels during differentiation of hematopoietic stem cells

  • Compare MAFK expression in normal versus dysplastic erythropoiesis

• Analyzing MAFK-NF-E2 interactions:

  • Apply co-immunoprecipitation with MAFK antibodies to isolate and study MAFK-NF-E2 complexes

  • Perform ChIP-seq to map binding sites of MAFK-NF-E2 heterodimers at globin gene loci

• Functional studies:

  • Combine MAFK knockdown/overexpression with protein detection using validated antibodies

  • Correlate MAFK protein levels with functional readouts of erythroid differentiation

• Diagnostic applications:

  • Develop immunohistochemistry protocols using MAFK antibodies to study bone marrow specimens

  • Investigate MAFK expression in hematological disorders

These approaches leverage MAFK antibodies to provide insights into the molecular mechanisms governing erythroid differentiation and potential therapeutic targets for hematological disorders .

What is the significance of MAFK detection in oxidative stress response studies?

MAFK and other small Maf proteins are implicated in cellular responses to oxidative stress, particularly through interactions with NRF2 (Nuclear factor erythroid 2-related factor 2). Researchers investigating oxidative stress can utilize MAFK antibodies to:

• Monitor MAFK expression changes under oxidative stress conditions:

  • Use Western blot or ELISA to quantify MAFK protein levels before and after oxidative stress induction

  • Apply flow cytometry to assess cell population heterogeneity in MAFK expression following stress

• Study MAFK-NRF2 interactions:

  • Employ co-immunoprecipitation with MAFK antibodies to isolate MAFK-NRF2 complexes

  • Perform ChIP-seq to identify genomic binding sites of MAFK-NRF2 complexes at antioxidant response elements (AREs)

• Investigate MAFK's dual roles:

  • Compare MAFK homodimer versus heterodimer formation under different redox conditions

  • Correlate MAFK protein levels with expression of antioxidant genes

• Develop therapeutic strategies:

  • Use MAFK antibodies to validate targets in drug screening approaches

  • Monitor MAFK expression as a biomarker for oxidative stress responses

Understanding MAFK's role in oxidative stress responses may provide insights into various pathological conditions including cancer, neurodegenerative diseases, and aging-related disorders.

How might single-cell protein analysis technologies enhance MAFK research?

Emerging single-cell technologies offer new opportunities for investigating MAFK expression and function:

• Single-cell Western blotting:

  • Apply MAFK antibodies in microfluidic platforms that enable Western blot analysis at the single-cell level

  • Investigate cell-to-cell variability in MAFK expression within heterogeneous populations

• Mass cytometry (CyTOF):

  • Develop metal-conjugated MAFK antibodies for high-dimensional protein profiling

  • Simultaneously measure MAFK expression alongside dozens of other proteins in single cells

• Imaging mass cytometry:

  • Combine MAFK antibody detection with spatial information in tissue contexts

  • Map MAFK expression patterns in relation to tissue architecture and cellular microenvironments

• Single-cell proteogenomics:

  • Integrate MAFK protein detection with single-cell transcriptomics

  • Correlate MAFK protein levels with gene expression profiles at the single-cell level

These approaches would provide unprecedented resolution of MAFK expression patterns and functional relationships, potentially revealing new insights into its role in transcriptional regulation and cellular function.

What are the promising developments in antibody technology that may improve MAFK detection?

Several emerging antibody technologies may enhance MAFK detection and research:

• Recombinant antibody fragments:

  • Single-chain variable fragments (scFvs) or nanobodies against MAFK epitopes

  • Smaller size allows better tissue penetration and potentially improved access to nuclear MAFK

• Site-specific labeling strategies:

  • Precisely controlled conjugation of fluorophores or other detection molecules

  • Enhanced signal-to-noise ratios and multiplexing capabilities

• Proximity-based detection systems:

  • Adaptation of technologies like SplitTurbo or HaloTag for enhanced detection of MAFK interactions

  • Improved sensitivity for detecting transient or weak protein-protein interactions

• CRISPR-based endogenous tagging:

  • Integration of epitope tags into the endogenous MAFK locus

  • Detection using highly validated tag antibodies, circumventing limitations of direct MAFK antibodies

• Antibody engineering for specific applications:

  • Development of antibodies optimized for live-cell imaging of MAFK

  • Engineering antibodies with improved performance in tissue clearing and 3D imaging applications

These technological advances may overcome current limitations in MAFK detection and enable new experimental approaches in studying its biological functions.