MAFB Antibody, Biotin conjugated

What is MAFB and what cellular functions does it regulate?

MAFB (V-maf musculoaponeurotic fibrosarcoma oncogene homolog B) is a transcription factor belonging to the large Maf family that shares similar basic region/leucine zipper DNA binding motifs and N-terminal activation domains. It functions as both a transcriptional activator and repressor, depending on cellular context . MAFB plays pivotal roles in:

• Regulating lineage-specific hematopoiesis by repressing ETS1-mediated transcription of erythroid-specific genes in myeloid cells

• Directing differentiation of monocytic cells, macrophages, osteoclasts, podocytes, and islet beta cells

• Supporting renal tubule survival and F4/80 maturation

• Activating insulin and glucagon promoters

• Acting as either an oncogene or tumor suppressor depending on cellular context

MAFB's expression is significantly elevated in response to metabolic and immunological stimuli that promote macrophage M2 polarization and cholesterol efflux, while being downregulated by pro-inflammatory pathogenic triggers .

What applications is the MAFB Antibody, Biotin conjugated most suitable for?

The biotin-conjugated MAFB antibody is primarily optimized for ELISA applications as indicated in the product information . The biotin conjugation allows for enhanced sensitivity through signal amplification using streptavidin-based detection systems. While ELISA is the validated application, other MAFB antibodies have been successfully used in:

• Western blotting (1:1000 dilution recommended)

• Immunoprecipitation (1:50 dilution recommended)

• Single-cell analysis (<0.5μg/test)

• Immunohistochemistry on paraffin or frozen sections

When adapting the biotin-conjugated antibody for applications beyond ELISA, validation experiments are essential to confirm specificity and optimal working conditions.

What is the recommended storage and handling protocol for maintaining antibody activity?

For optimal preservation of the MAFB antibody's activity, the following storage and handling protocols are recommended:

• Store at 2-8°C for up to one year after shipment

• The antibody is supplied in PBS with 1mM EDTA and 0.09% sodium azide as a preservative

• For the biotin-conjugated version, the standard storage buffer contains 50% Glycerol, 0.01M PBS, pH 7.4, and 0.03% Proclin 300 as a preservative

• Avoid repeated freeze-thaw cycles

• When working with the antibody, maintain cold chain practices

• Centrifuge briefly before opening to ensure all liquid is at the bottom of the vial

Long-term storage beyond one year may be possible at -20°C for some antibody formulations, but this should be validated for each specific lot.

How should researchers optimize immunohistochemical protocols for MAFB detection in different tissues?

Optimizing immunohistochemical protocols for MAFB detection requires consideration of tissue type and target cell populations. Based on established methods, the following protocol framework is recommended:

• Tissue preparation:

  • Fix tissues in 4% paraformaldehyde in phosphate-buffered saline overnight

  • Process for either paraffin embedding or frozen sections

  • Cut sections at 8-12 μm thickness

• Antibody incubation:

  • For paraffin sections: Perform heat-induced epitope retrieval

  • Block endogenous peroxidase with 0.3% H₂O₂

  • Use a 1:1,000 dilution of anti-MAFB antibody

  • Incubate overnight at 4°C

• Detection system:

  • For biotin-conjugated antibodies: Use streptavidin-HRP

  • Develop with DAB/H₂O₂ with or without 2.5% NiNH₄SO₄ (for black vs. brown reaction products)

  • For dual staining (e.g., MAFB and F4/80), distinguish immunoreactivity by brown (without NiNH₄SO₄) and black (with NiNH₄SO₄) reaction products

For renal tissue specifically, researchers should pay particular attention to podocyte staining, while for macrophage studies, co-staining with F4/80 can provide cellular context for MAFB expression.

What controls are essential when using biotin-conjugated antibodies in tissues with endogenous biotin?

When using biotin-conjugated antibodies in tissues with high endogenous biotin (such as kidney, liver, and brain), the following controls and blocking steps are essential:

• Endogenous biotin blocking:

  • Pretreat tissue sections with avidin followed by biotin before applying primary antibody

  • Commercial endogenous biotin blocking kits are available and recommended

• Essential controls:

  • Negative control: Omit primary antibody but include all other reagents

  • Endogenous biotin control: Treat section with detection system only, omitting primary antibody

  • Isotype control: Use biotin-conjugated rabbit IgG at the same concentration as the MAFB antibody

  • Positive control: Include tissue with known MAFB expression (e.g., renal podocytes or macrophages)

• Alternative approach:

  • Consider using a non-biotin detection system when working with biotin-rich tissues

  • For complex tissues, biotin-streptavidin blocking kits specifically designed for immunohistochemistry should be employed

Proper blocking and controls ensure that signals detected are specific to MAFB rather than endogenous biotin or non-specific binding.

How can MAFB antibodies be used to investigate its role in transcriptional regulation and oncogenic pathways?

Investigating MAFB's role in transcriptional regulation and oncogenic pathways requires sophisticated experimental approaches:

• Chromatin Immunoprecipitation (ChIP):

  • Use MAFB antibodies to identify genomic binding sites

  • Focus particularly on the G1 element of the glucagon promoter and ETS1-regulated genes

  • For biotin-conjugated antibodies, streptavidin magnetic beads can be used for pulldown

• Co-immunoprecipitation (Co-IP):

  • Investigate MAFB interactions with other transcription factors, particularly:

    • ETS2 (direct binding partner)

    • PCAF and P300 (co-activators)

    • Components of the Notch transcriptional complex

  • Recommended dilution for IP is 1:50

• Functional studies in leukemia models:

  • Use retroviral transduction/bone marrow transplant (BMT) models

  • Monitor CD4+CD8+ T cell populations in peripheral blood

  • Assess disease progression through WBC counts, spleen weights, and infiltrating tumor cells in tissues

  • Design experiments that measure Notch1 signaling intensity in the presence or absence of MAFB

• Oligo-immunoprecipitation (OIP) assays:

  • Use biotinylated oligonucleotides containing consensus Rbpj/Notch1 (CSL) binding sequences

  • Assess MAFB binding to these sequences in the presence/absence of ETS2

These techniques allow researchers to delineate MAFB's complex roles in both normal development and oncogenic transformation.

What are the considerations for incorporating MAFB antibody in single-cell analysis workflows?

Incorporating MAFB antibodies into single-cell analysis requires careful optimization and consideration of several factors:

• Antibody concentration and specificity:

  • Use at <0.5μg/test as recommended for single-cell applications

  • Validate specificity in your cell type of interest

  • Consider using conjugated antibodies compatible with flow cytometry or imaging platforms

• Integration with 10x Genomics platforms:

  • The antibody has been validated for use with "10x Genomics Gene Expression Flex with Feature Barcodes and Multiplexing product"

  • Follow the specific "MultiPro™ Cell Surface and Intracellular Staining Protocol" provided by manufacturers

  • For intracellular transcription factors like MAFB, ensure proper cell fixation and permeabilization

• Multi-parameter analysis:

  • Combine with other markers to identify specific cell populations:

    • Use F4/80 for macrophage identification

    • Use podocyte markers (nephrin, podocin) for kidney cells

    • Use lineage markers for hematopoietic populations

• Data analysis considerations:

  • Account for background and autofluorescence

  • Use proper compensation when multiplexing

  • Consider the bimodal distribution of transcription factors when setting gates

The 5CFLX oligonucleotide conjugate (Barcode Sequence: TCGCGGACCAGGAAT) allows for identification of MAFB-expressing cells in complex cell mixtures when using compatible platforms .

How can researchers troubleshoot unexpected results when using MAFB antibodies in Western blotting?

When troubleshooting MAFB antibody performance in Western blotting, researchers should consider:

• Molecular weight considerations:

  • Expected molecular weights: 36 kDa (calculated) versus 46-48 kDa (observed in some cell types)

  • This discrepancy could reflect post-translational modifications, particularly SUMO modification which controls MAFB's transcriptional activity

• Sample preparation optimization:

  • Use phosphatase inhibitors to preserve phosphorylated forms

  • Include SUMO protease inhibitors (such as N-ethylmaleimide) in lysis buffers

  • Optimize lysis conditions for nuclear proteins (MAFB is a transcription factor)

• Technical troubleshooting steps:

  Issue	Potential Solution
  No signal	Increase antibody concentration; verify protein transfer; check sample preparation
  Multiple bands	Validate with knockout/knockdown controls; consider isoforms or degradation products
  Higher MW than expected	Check for post-translational modifications; consider SUMO modification
  Lower MW than expected	Evaluate for protein degradation during sample preparation

• Positive controls:

  • Include cell lines with known MAFB expression (e.g., macrophages, osteosarcoma cell lines)

  • Consider using recombinant MAFB protein as a positive control

  • The immunogen used for antibody production was recombinant Human MAFB protein (168-323AA)

When optimizing Western blot conditions, start with the recommended 1:1000 dilution and adjust based on signal strength and background.

What methodologies are recommended for studying MAFB's role in macrophage polarization and tumor-associated macrophages?

MAFB is significantly involved in macrophage polarization and serves as a marker for tumor-associated macrophages (TAMs). The following methodologies are recommended:

• Macrophage polarization studies:

  • Use MAFB antibodies to track expression changes during M1/M2 polarization

  • Combine with flow cytometry for other polarization markers

  • Monitor MAFB expression in response to various stimuli that promote M2 polarization or cholesterol efflux

• Tumor-associated macrophage identification:

  • Perform dual immunostaining for MAFB and F4/80 in tumor sections

  • Use immunofluorescence with confocal microscopy for co-localization studies

  • Consider using the validated protocol: Incubate with anti-MAFB (1:1000 dilution) and anti-F4/80 (rat polyclonal, 1:1000) antibodies

• Functional studies:

  • Perform MAFB knockdown in macrophages using shRNA approaches

  • Assess impact on polarization markers, phagocytic capacity, and cytokine production

  • Evaluate changes in macrophage-tumor cell interactions following MAFB manipulation

• Single-cell analysis of tumor microenvironment:

  • Use the validated single-cell protocols (<0.5μg/test)

  • Integrate with RNA-seq data to correlate MAFB protein levels with transcriptional signatures

  • Apply clustering algorithms to identify MAFB-high macrophage subpopulations within the tumor microenvironment

MAFB is required for proliferation and tumorigenicity in certain cancer types and serves as a marker for TAMs in both mouse and human tumors , making these approaches valuable for cancer immunology research.

How can researchers effectively use MAFB antibodies to investigate its role in renal development and podocyte biology?

MAFB plays an essential role in renal development and podocyte biology. The following approaches are recommended for investigating these functions:

• Developmental studies:

  • Use MAFB antibodies for immunohistochemistry on embryonic kidney sections

  • Combine with TUNEL assays to assess apoptosis in MAFB-deficient models

  • Track podocyte development using co-staining with nephrin and podocin antibodies

• Podocyte-specific analysis:

  • Optimize immunostaining protocols for glomerular sections

  • Use paraffin or frozen sections (8-12 μm) with appropriate antigen retrieval

  • Employ double immunostaining with podocyte markers (nephrin, podocin) to confirm cell-specific expression

• Functional assays:

  • Investigate MAFB's role in podocyte differentiation through knockdown/knockout approaches

  • Monitor podocyte function (e.g., filtration) following MAFB manipulation

  • Assess the impact of MAFB on podocyte survival under stress conditions

• Protocol optimization:

  • For co-staining experiments, distinguish immunoreactivity using different chromogens:

    • Brown (DAB without NiNH₄SO₄) for one marker

    • Black (DAB with NiNH₄SO₄) for the second marker

  • For in situ hybridization combined with immunohistochemistry, follow protocols described in previous studies of podocyte development

These approaches will help elucidate MAFB's critical role in kidney development, particularly in podocyte differentiation and function, which are essential for proper renal filtration.

What experimental design should be considered when using MAFB antibodies to study its role in Notch signaling pathways?

MAFB enhances oncogenic Notch signaling in T-cell acute lymphoblastic leukemia (T-ALL). When designing experiments to investigate this role, researchers should consider:

The experimental approach should account for the synergistic relationship between MAFB and ETS factors, which together can amplify the output of weakly activating Notch1 mutants to levels comparable to those induced by core components of the Notch transcriptional complex.

What are the most common issues when using biotin-conjugated antibodies and how can they be resolved?

When working with biotin-conjugated MAFB antibodies, researchers may encounter several common issues. Here are solutions to address them:

For biotin-conjugated antibodies specifically, remember that:

• Biotin-rich tissues (kidney, liver, brain) require special blocking steps

• Streptavidin-HRP concentration may need optimization

• Signal amplification can increase both specific signal and background

• Some fixatives can affect biotin conjugation or accessibility

Always include appropriate controls and optimize each step of the protocol for your specific tissue or cell type of interest.

How should researchers approach validation of MAFB antibody specificity for their particular application?

Thorough validation of MAFB antibody specificity is crucial for reliable experimental results. Researchers should follow these comprehensive validation steps:

• Genetic controls:

  • Compare staining patterns in MAFB knockout/knockdown versus wild-type samples

  • Use siRNA or shRNA approaches to create transient knockdown controls

  • Consider the knockdown approach used in T-ALL studies with MafB shRNA

• Expression pattern validation:

  • Verify that staining patterns match known MAFB expression profiles:

    • Podocytes in kidney

    • Macrophages (particularly F4/80+ populations)

    • Islet beta cells

  • Compare results with published immunohistochemistry data

• Multi-technique confirmation:

  • Correlate protein detection with mRNA expression (RT-PCR, in situ hybridization)

  • Compare results across different detection methods (Western blot, immunohistochemistry, flow cytometry)

  • Verify that molecular weight matches expected size (36 kDa calculated, but 46-48 kDa observed due to modifications)

• Cross-reactivity assessment:

  • Test on tissues/cells known to be negative for MAFB

  • Consider potential cross-reactivity with other MAF family members

  • Use peptide competition assays to confirm epitope specificity

• Application-specific validation:

  Application	Validation Approach
  Western blot	Verify single band at expected MW; include positive control cell types
  IHC/IF	Compare with in situ hybridization patterns; perform peptide blocking
  ELISA	Generate standard curves with recombinant protein; determine LOD and dynamic range
  Single-cell	Compare to flow cytometry; validate with sorting and subsequent analysis

Remember that the immunogen used for antibody production was recombinant Human MAFB protein (168-323AA) , which should be considered when interpreting results.