MBIP Antibody

What is MBIP and what cellular functions does it perform?

MBIP (MAP3K12 binding inhibitory protein 1), also known as MAPK upstream kinase (MUK)-binding inhibitory protein, is a 344 amino acid nuclear and cytoplasmic protein that plays a crucial role in cellular signaling. It functions primarily by inhibiting MAP3K12 (ZPK) activity, which subsequently induces the activation of the JNK/SAPK pathway . MBIP is ubiquitously expressed throughout the body, with notably high expression levels in lung and heart tissues . Beyond its role in MAPK signaling, MBIP also serves as a component of the ATAC complex, which exhibits histone acetyltransferase activity specifically on histones H3 and H4 . This dual functionality positions MBIP at the intersection of cytoplasmic signaling and nuclear gene regulation processes.

What applications are MBIP antibodies validated for?

MBIP antibodies have been extensively validated for multiple research applications, with consistent performance across different manufacturers' products. The primary applications include Western Blot (WB), Immunoprecipitation (IP), Immunohistochemistry (IHC), and Enzyme-Linked Immunosorbent Assay (ELISA) . Some specialized MBIP antibodies also demonstrate reliability in Immunocytochemistry/Immunofluorescence (ICC/IF) and Flow Cytometry (intracellular) . When selecting an MBIP antibody, researchers should verify that the specific clone has been validated for their intended application, as performance can vary between experimental contexts. For instance, the rabbit recombinant monoclonal antibodies from Abcam (clones EPR13951 and EPR13952) have different application profiles, with EPR13951 being suitable for flow cytometry while EPR13952 is not specifically validated for this application .

What species reactivity do commercially available MBIP antibodies exhibit?

The species reactivity of MBIP antibodies varies by manufacturer and clone. Many commercially available MBIP antibodies demonstrate reactivity with human samples across all applications . Some antibodies, like Proteintech's 66102-1-Ig, also show cross-reactivity with mouse samples . This cross-species reactivity is particularly valuable for comparative studies between human and mouse models. When planning experiments involving other species, researchers should carefully review the manufacturer's validation data or consider performing preliminary validation tests to confirm antibody performance in their specific experimental system. The conservation of MBIP protein sequence across species can influence cross-reactivity, making some antibodies more versatile than others for comparative studies.

How do different MBIP antibody clones compare in specificity and sensitivity?

Multiple MBIP antibody clones are available commercially, each with distinct characteristics that may influence experimental outcomes. The mouse monoclonal antibody from Proteintech (66102-1-Ig) demonstrates broad reactivity across multiple human cell lines including HeLa, HEK-293, HepG2, LNCaP, Jurkat, THP-1, and K-562 . Biorbyt's mouse monoclonal clone 8G3 (orb395294) shows comparable reactivity profiles but with potentially different epitope recognition . For researchers requiring rabbit-derived antibodies, Abcam offers two recombinant monoclonal options: EPR13951 (ab186750) and EPR13952 (ab181040) .

Western blot data indicates that EPR13951 works effectively at higher dilutions (1/10000) while still maintaining strong signal intensity in both cell and tissue lysates . By comparison, EPR13952 is typically used at lower dilutions (1/1000) and has been specifically validated for immunoprecipitation studies . When selecting between these clones, researchers should consider not only the host species and applications but also the dilution ranges and sample types that have been validated. For critical experiments, direct comparison of multiple antibody clones may be warranted to identify the optimal reagent for specific experimental conditions.

What are the recommended protocols for optimizing MBIP antibody performance in Western blot applications?

For optimal Western blot results with MBIP antibodies, several technical factors should be considered. The recommended dilution ranges vary significantly between antibody clones: Proteintech's 66102-1-Ig works best at 1:3000-1:8000 , Biorbyt's orb395294 at 1:500-1:2000 , and Abcam's EPR13951 at 1:10000 . These differences highlight the importance of antibody titration for each experimental system.

Blocking conditions also influence performance, with 5% non-fat dry milk in TBST being effectively used with Abcam's antibodies . For detection systems, both HRP-conjugated anti-rabbit and anti-mouse secondary antibodies have been successfully employed, typically at dilutions around 1:1000 . Sample preparation is equally critical - Western blot validation data shows consistent MBIP detection across diverse sample types including cell lines (HeLa, HEK-293, HepG2, Caco-2) and tissue lysates (human fetal lung and heart) .

When troubleshooting, researchers should consider that the protein runs consistently at approximately 39 kDa, though some variation (up to 45 kDa) may occur depending on the gel system and sample type . For particularly challenging samples, optimizing protein loading (typically 10-20 μg of total protein) and extending primary antibody incubation times may improve signal intensity while maintaining specificity.

What factors influence MBIP detection in immunohistochemistry applications?

Successful immunohistochemical detection of MBIP requires careful consideration of several technical parameters. Antibody dilution represents a critical variable, with recommended ranges of 1:400-1:1600 for Proteintech's 66102-1-Ig and 1:20-1:200 for Biorbyt's orb395294 . These substantial differences emphasize the importance of antibody titration for each experimental system.

Antigen retrieval methodology significantly impacts MBIP epitope accessibility in fixed tissues. For Proteintech's antibody, TE buffer at pH 9.0 is recommended as the primary retrieval method, though citrate buffer at pH 6.0 provides an alternative approach . The effectiveness of these retrieval methods may vary depending on tissue type and fixation conditions. Positive detection has been documented in several tissue types including human kidney and mouse testis , as well as human heart tissue .

When optimizing IHC protocols, researchers should consider fixation duration, section thickness, retrieval time, and primary antibody incubation conditions. For challenging tissues, extending antibody incubation times or implementing signal amplification systems may improve detection sensitivity without compromising specificity. The subcellular localization pattern observed should be consistent with MBIP's known distribution in both nuclear and cytoplasmic compartments, serving as an internal validation measure.

What are the best practices for MBIP immunoprecipitation experiments?

Immunoprecipitation (IP) of MBIP requires careful optimization of antibody-to-lysate ratios and buffer conditions. For Proteintech's 66102-1-Ig, the recommended protocol uses 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate . Biorbyt's orb395294 is suggested at a dilution of 1:200-1:1000 for IP applications . Abcam's EPR13952 (ab181040) has been specifically validated for IP applications with demonstrated success in 293T cell lysates .

When developing an IP protocol, consider that MBIP has been successfully immunoprecipitated from HEK-293 cells , making this cell line a reliable starting point for protocol optimization. Western blot analysis of IP pellets confirms that the immunoprecipitated protein consistently appears at the expected molecular weight of approximately 39 kDa . For co-immunoprecipitation studies investigating MBIP's interaction with other proteins, buffer composition becomes particularly critical - mild lysis conditions that preserve protein-protein interactions should be employed.

To minimize non-specific binding, pre-clearing lysates with appropriate control IgG and protein A/G beads is recommended. Including appropriate negative controls, such as immunoprecipitation with non-specific IgG or using 1XPBS instead of cell lysate, is essential for confirming specificity . For researchers investigating MBIP's interaction with components of the ATAC complex or proteins in the JNK pathway, co-IP protocols may need further refinement to preserve potentially transient interactions.

How can researchers validate MBIP antibody specificity in their experimental systems?

Validating MBIP antibody specificity is essential for generating reliable research data. A multi-faceted approach to validation is recommended, beginning with molecular weight verification. The expected molecular weight of MBIP is approximately 39 kDa, though some variation (up to 45 kDa) has been reported . Consistent band patterns across multiple cell lines and tissues provide initial evidence of specificity .

For definitive validation, genetic approaches offer the highest level of confidence. These include:

• siRNA or shRNA knockdown, which should result in diminished signal intensity

• CRISPR/Cas9 knockout, which should completely eliminate specific antibody binding

• Overexpression systems, which should produce increased signal intensity at the expected molecular weight

Peptide competition assays, where pre-incubation of the antibody with the immunizing peptide blocks specific binding, provide another validation approach. Cross-validation using multiple antibodies targeting different MBIP epitopes can further confirm specificity - if multiple antibodies show consistent patterns, specificity is more likely.

For immunohistochemistry and immunofluorescence applications, comparing the observed localization pattern with published subcellular distribution data provides additional validation. MBIP's known nuclear and cytoplasmic distribution should be reflected in the staining pattern . Finally, positive and negative control samples with known MBIP expression levels should be included in all experiments to establish baseline expectations for antibody performance.

How does MBIP function in the JNK/SAPK signaling pathway and how can antibodies help investigate this role?

MBIP inhibits MAP3K12 (also known as ZPK) activity, which leads to the activation of the JNK/SAPK pathway . This inhibitory function positions MBIP as a critical regulator of stress-responsive signaling cascades. Investigating this regulatory relationship requires experimental approaches that can detect both protein-protein interactions and functional outcomes. Co-immunoprecipitation experiments using MBIP antibodies can capture and identify interaction partners within the JNK pathway. Western blot analysis with phospho-specific antibodies for JNK/SAPK pathway components can then assess the functional consequences of MBIP modulation.

To comprehensively study MBIP's role in JNK signaling, researchers might consider combinatorial approaches such as proximity ligation assays to visualize MBIP-MAP3K12 interactions in situ, or MBIP knockdown/overexpression paired with measurements of downstream JNK activation. Cell stress experiments (UV irradiation, osmotic shock, or inflammatory cytokine exposure) can reveal how MBIP influences stress-induced JNK activation. The availability of multiple validated MBIP antibodies enables these diverse experimental approaches, allowing researchers to investigate both the physical interactions and functional consequences of MBIP in JNK/SAPK signaling networks.

What role does MBIP play in the ATAC complex and how can researchers study this function?

MBIP functions as a component of the ATAC complex, which exhibits histone acetyltransferase activity specifically on histones H3 and H4 . This places MBIP at the intersection of signaling pathways and epigenetic regulation. To investigate MBIP's role in the ATAC complex, researchers can employ chromatin immunoprecipitation (ChIP) experiments using MBIP antibodies to identify genomic regions where MBIP-containing complexes bind. Sequential ChIP (Re-ChIP) with antibodies against other ATAC complex components can confirm co-localization of these factors at specific genomic loci.

Co-immunoprecipitation studies with MBIP antibodies followed by mass spectrometry analysis can identify all protein interactions within the ATAC complex and potentially reveal novel associations. For functional studies, MBIP depletion combined with histone acetylation assays (Western blot with acetyl-H3/H4 antibodies or ChIP-seq for acetylation marks) can determine how MBIP contributes to ATAC complex activity. Immunofluorescence co-localization studies using MBIP antibodies alongside other ATAC component antibodies can visualize complex formation in different cellular compartments and under various physiological conditions. These approaches leverage the specificity of MBIP antibodies to dissect both the structural organization and functional significance of MBIP within the ATAC complex.

How can MBIP antibodies be optimized for flow cytometry applications?

While not all MBIP antibodies are validated for flow cytometry, Abcam's EPR13951 (ab186750) has been specifically tested for intracellular flow cytometry applications . Optimizing MBIP detection by flow cytometry requires careful attention to several technical parameters. Fixation and permeabilization methods significantly impact intracellular epitope accessibility - for most applications, paraformaldehyde fixation (2-4%) followed by permeabilization with either saponin (0.1-0.5%) or Triton X-100 (0.1%) has proven effective for intracellular proteins.

Antibody titration is essential, with initial testing recommended at the manufacturer's suggested dilution followed by serial dilutions to identify optimal signal-to-noise ratios. Appropriate blocking steps using serum matched to the secondary antibody species can minimize non-specific binding. For multi-parameter flow cytometry, careful selection of fluorophores with minimal spectral overlap enables simultaneous detection of MBIP with other proteins of interest.

Control samples are critical for setting appropriate gates and confirming specificity - these should include unstained cells, secondary-only controls, and ideally cells with genetically manipulated MBIP expression levels. When analyzing flow cytometry data for MBIP, researchers should consider that as both a nuclear and cytoplasmic protein, MBIP may show particular fixation/permeabilization dependencies that affect signal intensity and population distribution in flow histograms.

What are the tissue-specific expression patterns of MBIP and appropriate controls for immunohistochemistry?

MBIP demonstrates ubiquitous expression across tissues, with notably higher expression levels reported in lung and heart tissues . This expression pattern provides important context for immunohistochemical studies, as staining intensity should correlate with known expression levels. Validated positive control tissues for MBIP immunohistochemistry include human kidney, mouse testis, and human heart . These tissues have shown consistent positive staining with multiple MBIP antibody clones and can serve as reliable controls for protocol optimization.

When performing IHC studies, tissue-specific considerations become important. Antigen retrieval methods may need optimization for different tissues - while TE buffer at pH 9.0 is generally recommended, citrate buffer at pH 6.0 may prove more effective for certain tissue types . Appropriate negative controls should include both technical controls (primary antibody omission, isotype control antibody substitution) and biological controls (tissues with known low MBIP expression).

For comparative studies examining MBIP expression across multiple tissues or disease states, standardization of staining protocols becomes crucial to ensure that observed differences reflect genuine biological variation rather than technical artifacts. Quantification of MBIP staining should consider both the intensity and pattern of expression, as subcellular localization may vary in different tissue contexts or disease states, potentially reflecting functional adaptation of MBIP in diverse cellular environments.

What are common issues encountered when using MBIP antibodies and how can they be resolved?

Researchers working with MBIP antibodies may encounter several common technical challenges. In Western blot applications, weak or absent signals might result from insufficient protein loading, suboptimal antibody dilution, or inadequate transfer. Increasing protein load (20 μg typically works well) , adjusting antibody concentration based on manufacturer recommendations , and optimizing transfer conditions for proteins in the 39-45 kDa range can address these issues.

Non-specific bands may appear due to insufficient blocking or excessive antibody concentration. Implementing more stringent blocking conditions (5% NFDM/TBST has proven effective) and further diluting the primary antibody can improve specificity. For immunohistochemistry, high background staining often results from inadequate blocking or excessive antibody concentration. Extending blocking steps, using alternative blocking agents, and diluting antibodies according to validated ranges can enhance signal-to-noise ratios.

Poor or inconsistent immunoprecipitation results may stem from suboptimal antibody-to-lysate ratios. Following the validated ratios (0.5-4.0 μg antibody per 1.0-3.0 mg lysate) and including appropriate controls helps troubleshoot these issues. Batch-to-batch variation between antibody lots can also impact experimental reproducibility. Implementing rigorous internal validation protocols for each new antibody lot, including side-by-side comparison with previously verified lots, helps maintain consistent experimental outcomes across studies.

How should researchers validate experimental findings across different MBIP antibody clones?

Cross-validation using multiple antibody clones targeting different MBIP epitopes provides robust confirmation of experimental findings. When planning cross-validation studies, researchers should select antibodies from different manufacturers or clones recognizing distinct regions of the MBIP protein. For example, comparing results between mouse monoclonal antibodies like Proteintech's 66102-1-Ig or Biorbyt's orb395294 with rabbit monoclonal antibodies such as Abcam's EPR13951 or EPR13952 can provide strong validation.

The cross-validation process should include identical experimental conditions for all antibodies, with side-by-side performance of key experiments like Western blot, immunohistochemistry, or immunoprecipitation. Results should demonstrate consistent molecular weight detection, similar expression patterns across tissues or cell lines, and comparable subcellular localization profiles. Significant discrepancies between antibody clones warrant further investigation to determine which provides the most specific detection.

For critical findings, orthogonal validation approaches that do not rely on antibodies (such as mRNA quantification, mass spectrometry, or genetic manipulation) provide additional confidence. Documentation of validation experiments, including the specific antibody clones, catalog numbers, and experimental conditions used, should be included in research publications to enhance reproducibility and transparency. This multi-antibody, multi-method approach to validation represents the gold standard for generating reliable data on MBIP expression and function.

What are the future directions for MBIP research and antibody development?

As MBIP research progresses, several promising directions emerge for both basic science and antibody technology development. The dual functionality of MBIP in signaling regulation (through MAP3K12 inhibition) and epigenetic processes (via the ATAC complex) positions it as a potential integrator of cellular stress responses and gene regulation . Future research may focus on how these distinct functions are coordinated and regulated in different cellular contexts or disease states.

From a technological perspective, the development of more specialized MBIP antibody tools could significantly advance the field. These might include phospho-specific antibodies targeting potential MBIP modification sites, conformation-specific antibodies that distinguish between different functional states, and antibodies suitable for super-resolution microscopy to precisely localize MBIP within subcellular structures. Expanding the species cross-reactivity of available antibodies would facilitate comparative studies across model organisms.