MIB2 Antibody, FITC conjugated

What is MIB2 and why is it important in cell signaling research?

MIB2 (Mind Bomb 2) is an E3 ubiquitin ligase that plays a critical role in regulating inflammatory signaling through NF-κB pathways. The protein contains five conserved domains: two MIB/Herc domains, an ankyrin repeat domain, and two RING domains that are essential for its catalytic activity . MIB2 has gained significant research attention due to its involvement in inflammatory responses through the ubiquitin-dependent degradation of CYLD, a deubiquitinating enzyme that negatively regulates NF-κB signaling . Studies using knockout models have demonstrated that MIB2 deficiency leads to reduced inflammatory responses, as evidenced by decreased serum interleukin-6 (IL-6) levels and suppressed inflammation in arthritis models . This makes MIB2 an important target for understanding inflammatory mechanisms and potentially developing therapeutic approaches for inflammatory diseases.

What are the key specifications of the FITC-conjugated anti-MIB2 antibody?

The FITC-conjugated anti-MIB2 antibody is designed to target amino acids 546-784 of the MIB2 protein, corresponding to a region that contains functional domains critical for MIB2 activity . This antibody is:

• Raised in rabbit as a polyclonal IgG antibody

• Affinity purified to ensure specificity

• Reactive against human MIB2 protein

• Suitable for Western blotting (WB) and immunoprecipitation (IP) applications

• Generated against recombinant MIB2 (Val546-Asn784) expressed in E. coli

• Available in 100 μg quantities

The FITC conjugation provides direct fluorescence visualization capability, eliminating the need for secondary antibody incubation in certain applications, which can be particularly advantageous for multicolor immunofluorescence studies.

How does the FITC conjugation affect antibody performance compared to unconjugated versions?

The FITC conjugation to the anti-MIB2 antibody provides direct visualization capability through green fluorescence emission when excited at appropriate wavelengths. While this conjugation offers significant advantages for certain applications, researchers should consider several performance aspects:

The FITC conjugation may slightly alter binding kinetics compared to unconjugated versions, potentially requiring optimization of antibody concentration in experimental protocols . The same antibody is also available in unconjugated form or with biotin conjugation, providing flexibility for different experimental designs . FITC has a relatively high photobleaching rate compared to some newer fluorophores, which may affect long-term imaging studies. For quantitative applications, researchers should account for potential signal loss over time and implement appropriate controls .

The FITC-conjugated antibody eliminates potential cross-reactivity issues from secondary antibodies but may have reduced signal amplification compared to two-step detection methods using unconjugated primary antibodies.

How can the FITC-conjugated MIB2 antibody be used to investigate MIB2-CYLD interactions?

The FITC-conjugated MIB2 antibody provides a powerful tool for investigating the critical interaction between MIB2 and CYLD, which forms the basis of MIB2's inflammatory regulation function. Research has established that MIB2 interacts with CYLD primarily through its ankyrin repeat domain, while CYLD binds to MIB2 via its third CAP domain (amino acids 287-589) .

For co-localization studies, researchers can use the FITC-conjugated MIB2 antibody alongside a differently labeled CYLD antibody (e.g., with a red fluorophore) to visualize their spatial relationship in cellular contexts. Immunofluorescence studies have demonstrated that both proteins co-localize in the cytoplasm, which is consistent with their functional interaction .

For interaction validation studies, the antibody can be employed in co-immunoprecipitation experiments where cell lysates are immunoprecipitated with the MIB2 antibody followed by immunoblotting for CYLD, or vice versa. Previous research has successfully demonstrated this interaction using endogenous proteins in HEK293T cells through immunoprecipitation with an anti-MIB2 antibody .

For domain mapping experiments, the antibody can be used in conjunction with truncated forms of MIB2 or CYLD to confirm which specific domains mediate their interaction, complementing previous findings using AlphaScreen assays and cell-based methods.

What role does MIB2 play in NF-κB signaling and how can the antibody help elucidate this pathway?

MIB2 enhances inflammatory signaling through a specific mechanistic pathway involving CYLD degradation and subsequent NF-κB activation. The FITC-conjugated MIB2 antibody can help investigate this pathway through several experimental approaches:

MIB2 catalyzes Lys-48-linked polyubiquitination of CYLD at Lys-338 and Lys-530, targeting it for proteasomal degradation . Using the antibody in ubiquitination assays can help visualize this process, especially when combined with proteasome inhibitors to accumulate ubiquitinated species.

The degradation of CYLD by MIB2 activates NF-κB signaling through TNFα stimulation and the linear ubiquitination assembly complex (LUBAC) . The antibody can be used in time-course experiments following TNFα stimulation to track MIB2 expression, localization changes, and correlation with downstream signaling events.

A specific experimental protocol involves:

• Treating cells with TNFα at various time points

• Fixing and staining with FITC-conjugated MIB2 antibody

• Co-staining for NF-κB components or phosphorylated IκB

• Analyzing correlation between MIB2 expression/localization and NF-κB activation

Studies in MIB2-knockout models have shown reduced serum IL-6 and suppressed inflammatory responses in arthritis models , suggesting that antibody-based detection of MIB2 in animal tissue samples could provide valuable insights into inflammatory disease progression.

How can researchers use the MIB2 antibody to study ubiquitination processes?

The FITC-conjugated MIB2 antibody serves as a valuable tool for studying the complex ubiquitination processes mediated by this E3 ligase:

For ubiquitination site identification, the antibody can be used in conjunction with site-directed mutagenesis of potential ubiquitination sites on target proteins. Research has already identified that MIB2 catalyzes Lys-48-linked polyubiquitination of CYLD specifically at Lys-338 and Lys-530 .

An experimental approach involves:

• Generating CYLD mutants (K338R, K530R, or double mutants)

• Co-expressing with MIB2 in cell culture

• Immunoprecipitating with the MIB2 antibody

• Blotting for ubiquitin to compare ubiquitination levels

For ubiquitin chain-type determination, the antibody can be used in conjunction with specific antibodies against different ubiquitin linkages. Previous studies have determined that MIB2 predominantly catalyzes Lys-48-linked polyubiquitination rather than Lys-63-linked chains on CYLD . This type of ubiquitination typically targets proteins for proteasomal degradation, consistent with the observed decrease in CYLD protein levels when MIB2 is overexpressed.

To study the dynamic relationship between ubiquitination and protein stability, the antibody can be used in cycloheximide chase experiments to track MIB2 and its substrates over time. Such experiments have revealed that wild-type MIB2, but not catalytically inactive mutants, decreases the half-life of CYLD protein .

What are the optimal conditions for using FITC-conjugated MIB2 antibody in immunofluorescence studies?

Based on established protocols with similar FITC-conjugated antibodies, the following conditions are recommended for optimal immunofluorescence studies:

These conditions are similar to those used in previous studies where MIB2 was detected in U266 human myeloma cell line using a different MIB2 antibody at 10 μg/mL for 3 hours at room temperature . As MIB2 primarily localizes to the cytoplasm, appropriate counterstaining and imaging settings should be optimized to visualize this compartment clearly.

How should Western blotting protocols be optimized for MIB2 detection?

Effective Western blotting for MIB2 detection requires specific considerations:

When studying MIB2-mediated ubiquitination, consider including MG132 (proteasome inhibitor) treatment to stabilize ubiquitinated proteins. Additionally, include both wild-type and catalytically inactive MIB2 controls to verify specificity of ubiquitination signals .

What experimental design is optimal for studying MIB2-mediated protein degradation?

To effectively investigate MIB2's role in protein degradation, particularly of CYLD, the following experimental approach is recommended:

For cycloheximide chase assays:

• Transfect cells with target protein (e.g., CYLD) along with either wild-type MIB2, catalytically inactive MIB2 mutant, or empty vector

• Treat with cycloheximide (50-100 μg/mL) to inhibit new protein synthesis

• Collect cells at various time points (0, 2, 4, 6, 8 hours)

• Perform Western blotting with appropriate antibodies

• Quantify protein levels and calculate half-life

This approach has successfully demonstrated that wild-type MIB2, but not catalytically inactive mutants, decreases the half-life of CYLD protein .

For site-specific ubiquitination analysis:

• Generate mutants of the substrate protein at potential ubiquitination sites (e.g., CYLD-K338R, CYLD-K530R)

• Co-express with MIB2 and ubiquitin constructs

• Perform ubiquitination assays using immunoprecipitation

• Analyze with specific antibodies against different ubiquitin linkages

Previous research has established that MIB2 primarily catalyzes Lys-48-linked polyubiquitination rather than Lys-63-linked chains on CYLD , demonstrating the value of this approach.

What are common issues with FITC-conjugated antibodies and how can they be resolved?

Researchers working with FITC-conjugated MIB2 antibodies may encounter several challenges:

FITC has excitation/emission maxima around 495/519 nm, which can overlap with cellular autofluorescence. When designing multi-color experiments, pair FITC with fluorophores in distinctly different spectral ranges (e.g., far-red fluorophores) to minimize bleed-through .

How should researchers interpret complex data from MIB2-CYLD interaction studies?

The interpretation of MIB2-CYLD interaction data requires careful consideration of several factors:

When analyzing co-localization data, quantitative approaches such as Pearson's correlation coefficient or Manders' overlap coefficient should be used rather than subjective visual assessment. The expected pattern is cytoplasmic co-localization, as both MIB2 and CYLD have been shown to localize predominantly in this compartment .

For protein expression studies, it's critical to recognize that MIB2 overexpression should lead to decreased CYLD levels due to enhanced degradation. This inverse relationship is a key indicator of functional activity. When interpreting these results, researchers should:

• Compare CYLD levels in the presence of wild-type MIB2 vs. catalytically inactive MIB2

• Verify with proteasome inhibitors (e.g., MG132) to confirm degradation pathway

• Assess ubiquitination status with specific antibodies against Lys-48-linked polyubiquitin

When contradictory results emerge, consider:

• Cell type-specific effects (different cell lines may have varying levels of ubiquitination machinery)

• Transfection efficiency and expression levels of constructs

• Potential regulatory mechanisms affecting MIB2 activity

• Technical variations in antibody lots or experimental conditions

What controls are essential for validating MIB2 antibody specificity in research applications?

Proper controls are critical for ensuring the validity of research findings using MIB2 antibodies:

For siRNA knockdown validation, researchers should include:

• Non-targeting siRNA control

• MIB2-specific siRNA

• Rescue with siRNA-resistant MIB2 construct

This approach has been successfully used to validate the specificity of MIB2 antibodies and the functional effects of MIB2 depletion . When interpreting knockdown results, researchers should consider that incomplete knockdown may still allow sufficient MIB2 function, particularly if the protein has a long half-life or high catalytic efficiency.

How might the FITC-conjugated MIB2 antibody be used to investigate inflammatory diseases?

The FITC-conjugated MIB2 antibody presents significant potential for investigating inflammatory disease mechanisms, particularly given MIB2's established role in NF-κB signaling and inflammation:

For arthritis research, the antibody could be employed to examine MIB2 expression and localization in synovial tissues, building on findings that MIB2-knockout mice exhibit suppressed inflammatory responses in the K/BxN serum-transfer arthritis model . A potential experimental design would involve:

• Collecting synovial tissue samples from arthritis patients and controls

• Staining sections with FITC-conjugated MIB2 antibody alongside markers for inflammatory cells

• Quantifying MIB2 expression levels and correlating with disease severity

For studying inflammatory skin conditions, researchers could investigate the relationship between MIB2 and multiple familial trichoepitheliomas (MFT), as MIB2 has been shown to enhance degradation of a CYLD P904L variant identified in an MFT patient . The FITC-conjugated antibody would enable visualization of MIB2 distribution in skin biopsies and its co-localization with mutant CYLD variants.

Therapeutic development research could utilize the antibody to screen for compounds that modulate MIB2-CYLD interactions, potentially identifying novel anti-inflammatory agents that target this specific pathway.

What are emerging applications for studying MIB2 in cellular contexts beyond inflammation?

While MIB2's role in inflammatory pathways is well-established, emerging research suggests broader cellular functions that could be investigated using the FITC-conjugated antibody:

For subcellular trafficking studies, the antibody could track MIB2 localization under different cellular stresses or stimuli. Interestingly, research has shown that deletion of the RING domains of MIB2 (MIB2ΔRING) alters its localization from cytoplasmic to nuclear , suggesting potential unexplored nuclear functions that could be investigated using live-cell imaging with the FITC-conjugated antibody.

For protein-protein interaction networks, the antibody could be used in proximity ligation assays to identify novel MIB2 binding partners beyond CYLD, potentially uncovering new regulatory pathways.

In cancer research, the antibody could investigate potential dysregulation of MIB2 expression or function, particularly in myeloma where MIB2 expression has been detected in U266 human myeloma cells . The relationship between MIB2-mediated ubiquitination and cancer progression represents an underexplored area that could yield valuable insights into disease mechanisms.

How can researchers integrate the FITC-conjugated MIB2 antibody into multi-omics approaches?

The integration of antibody-based detection methods with other omics technologies offers powerful opportunities for comprehensive understanding of MIB2 biology:

For proteomics integration, researchers can:

• Use the antibody for immunoprecipitation followed by mass spectrometry to identify MIB2 interactors

• Combine with phosphoproteomics to reveal how MIB2-mediated signaling affects phosphorylation networks

• Implement Ubiquitin-remnant profiling to globally identify substrates affected by MIB2 activity

For transcriptomics correlation, researchers can analyze how MIB2 expression patterns (detected by the antibody) correlate with gene expression profiles in different cellular contexts or disease states, particularly focusing on NF-κB target genes.

For spatial biology applications, the FITC-conjugated antibody could be incorporated into multiplexed imaging approaches like Imaging Mass Cytometry or CODEX to visualize MIB2 distribution in relation to numerous other proteins simultaneously within tissue sections, providing unique insights into its spatial context in complex tissues and disease environments.