MIB2 Antibody, Biotin conjugated

What is MIB2 and what are its key biological functions?

MIB2 (Mind bomb homolog 2) is an E3 ubiquitin-protein ligase that plays critical roles in cellular signaling pathways. It contains multiple functional domains including RING finger domains and ankyrin repeats that facilitate protein-protein interactions and ubiquitination activities. MIB2 is primarily known for:

• Enhancing NF-κB activation through ubiquitin-dependent pathways

• Mediating degradation of CYLD (a deubiquitinating enzyme) through Lys-48-linked polyubiquitination

• Contributing to inflammatory responses through TNFα stimulation and the linear ubiquitination assembly complex (LUBAC)

• Potentially playing a role in NOTCH signaling pathways associated with cardiac development

Research has demonstrated that MIB2 directly interacts with CYLD via its ankyrin repeat region, catalyzing Lys-48-linked polyubiquitination at specific lysine residues (Lys-338 and Lys-530), which leads to CYLD degradation and subsequent activation of NF-κB signaling .

What experimental applications are suitable for MIB2 Antibody, Biotin conjugated?

MIB2 Antibody, Biotin conjugated is particularly valuable for several experimental applications in molecular and cellular biology research:

When performing IHC applications, researchers have successfully used MIB2 antibodies diluted at 1:600 for staining paraffin-embedded human skeletal muscle tissue, with visualization using HRP-conjugated streptavidin systems .

How does biotin conjugation enhance antibody detection compared to other labeling methods?

Biotin conjugation provides several distinct advantages for antibody-based detection methods:

The biotin-streptavidin interaction represents one of the strongest non-covalent biological interactions known (Kd ≈ 10^-15 M), providing exceptional stability during experimental procedures. Multiple biotin molecules can be conjugated to a single antibody molecule, allowing for significant signal amplification when detected with streptavidin-conjugated reporter molecules.

For detecting low-abundance proteins, biotin-labeled secondary antibodies can be combined with various streptavidin- or avidin-based conjugates (fluorescent, enzymatic) to achieve signal amplification that may not be possible with directly labeled antibodies. This approach permits detection of proteins expressed at levels below the threshold of conventional methods .

Additionally, biotinylated antibodies offer excellent versatility, as the same primary biotin-conjugated antibody can be paired with different streptavidin conjugates (HRP, fluorophores, gold particles) for multiple detection modalities without requiring different specifically-labeled antibodies.

What detection systems are compatible with MIB2 Antibody, Biotin conjugated?

MIB2 Antibody, Biotin conjugated can be utilized with multiple detection systems:

• Enzymatic Systems: Streptavidin-HRP conjugates for colorimetric detection in IHC and Western blotting

• Fluorescence Systems: Streptavidin-fluorophore conjugates (Alexa Fluor series) for immunofluorescence microscopy

• Tyramide Signal Amplification: Systems like the Biotin XX Tyramide SuperBoost Kit for enhanced sensitivity

• Flow Cytometry: Compatible with streptavidin-fluorophore conjugates for cell analysis

For maximal signal amplification, the Biotin XX Tyramide SuperBoost Kit with HRP-conjugated streptavidin can be employed, followed by detection with Alexa Fluor-streptavidin conjugates. This approach has been successfully demonstrated for detecting low-abundance proteins in cellular systems .

How does MIB2 regulate inflammatory signaling through CYLD degradation?

MIB2 plays a critical role in regulating inflammatory signaling through a specific mechanism involving CYLD degradation:

MIB2 interacts directly with CYLD through its ankyrin repeat region, which binds to the third CAP domain of CYLD. This interaction enables MIB2 to catalyze Lys-48-linked polyubiquitination of CYLD specifically at Lys-338 and Lys-530 residues. This polyubiquitination marks CYLD for proteasomal degradation, effectively reducing CYLD protein levels and stability. Since CYLD functions as a negative regulator of NF-κB signaling, its degradation consequently enhances NF-κB activation .

In vivo evidence supporting this mechanism comes from Mib2-knockout mice, which exhibit reduced serum interleukin-6 (IL-6) levels and suppressed inflammatory responses in arthritis models. This suggests that MIB2-mediated CYLD degradation is physiologically relevant in inflammatory conditions .

Notably, MIB2 does not use Lys-63-linked ubiquitination for CYLD, despite MIB2's reported ability to perform auto-ubiquitination through Lys-63 linkages. This specificity in ubiquitin chain type highlights the precision of MIB2's regulatory mechanism .

What optimization strategies improve MIB2 Antibody, Biotin conjugated performance in IHC applications?

Optimizing MIB2 Antibody, Biotin conjugated for IHC requires attention to several critical parameters:

Antigen Retrieval Protocol:
For paraffin-embedded tissues, high-pressure citrate buffer (pH 6.0) antigen retrieval has proven effective for MIB2 detection. This method helps expose epitopes that may be masked during fixation and embedding processes.

Blocking Protocol:
Implementation of a 30-minute room temperature blocking step with 10% normal goat serum effectively reduces non-specific binding. This is particularly important with biotin-conjugated antibodies due to potential background from endogenous biotin.

Antibody Incubation:
Optimal results have been achieved with overnight incubation at 4°C using the antibody diluted in 1% BSA solution. This extended incubation period enhances specific binding while minimizing background.

Detection System:
A biotinylated secondary antibody followed by HRP-conjugated streptavidin provides excellent visualization. For tissues with high levels of endogenous biotin (such as liver, kidney), consider using an Endogenous Biotin-Blocking Kit prior to antibody application .

Signal Amplification:
For detecting low-abundance MIB2 protein, implementing tyramide signal amplification through the Biotin XX Tyramide SuperBoost system can significantly enhance sensitivity without increasing background .

How can researchers validate MIB2 Antibody, Biotin conjugated specificity for experimental systems?

Ensuring antibody specificity is critical for generating reliable research data. For MIB2 Antibody, Biotin conjugated, the following validation approaches are recommended:

Positive Control Validation:
Human skeletal muscle tissue has been confirmed as an appropriate positive control for MIB2 antibody testing. Confirming expected staining patterns in this tissue provides a baseline for antibody performance .

Knockout/Knockdown Controls:
Utilization of MIB2 siRNA knockdown samples or Mib2-knockout mouse tissues provides the most stringent specificity control. Published data demonstrates that MIB2 antibody signals should significantly decrease in cells treated with MIB2-specific siRNA .

Peptide Competition:
Pre-incubating the antibody with recombinant MIB2 protein (specifically the immunogen fragment corresponding to amino acids 1-128) should abolish specific staining.

Western Blot Correlation:
Parallel Western blotting can confirm antibody specificity by detecting bands of expected molecular weight (~100 kDa for MIB2) that disappear in knockdown samples.

Multiple Antibody Concordance:
Comparing staining patterns with other validated MIB2 antibodies that recognize different epitopes can provide additional confirmation of specificity.

What technical considerations apply when using MIB2 Antibody, Biotin conjugated to investigate MIB2's role in inflammation models?

When investigating MIB2's role in inflammation with biotin-conjugated antibodies, researchers should consider:

Model Selection:
The K/BxN serum-transfer arthritis model has been validated for studying MIB2's role in inflammation. In this model, Mib2-knockout mice exhibited suppressed inflammatory responses and reduced serum IL-6 levels, making it an excellent system for investigating MIB2 function .

Stimulation Conditions:
TNFα stimulation activates the MIB2-dependent NF-κB signaling pathway. When designing experiments, researchers should carefully calibrate TNFα concentrations and exposure times to detect relevant changes in MIB2-dependent responses.

Quantification Methods:
Measurement of pro-inflammatory cytokines (particularly IL-6) provides a reliable readout of MIB2 activity in inflammatory contexts. ELISA or cytometric bead array methods can quantify these downstream effects.

Protein Stability Consideration:
When analyzing MIB2 protein levels or interactions, protein degradation inhibitors (such as MG132) may be necessary to prevent rapid turnover of MIB2 or its targets during experimental manipulation.

Interaction Studies:
When investigating MIB2-CYLD interactions, researchers have successfully employed multiple complementary approaches including AlphaScreen assays, GST-pulldown experiments, and co-immunoprecipitation with AGIA-tagged constructs. These methods provide reliable detection of MIB2-CYLD complex formation .

How do MIB2 variants affect protein functionality, and how can they be studied with antibody-based approaches?

MIB2 variants have been associated with significant phenotypic consequences, including cardiac abnormalities and Ménétrier-like gastropathy. Studying these variants requires specific approaches:

Functional Ubiquitination Assays:
In vitro functional ubiquitination assays have been used to assess the impact of MIB2 variants on protein activity. For example, the V742G variant (located in an ankyrin repeat domain) demonstrated reduced auto-ubiquitination compared to wild-type MIB2, suggesting impaired ubiquitin ligase activity .

Protein Stability Assessment:
Western blotting with MIB2 antibodies can reveal whether variants affect protein stability. The V984L variant, for instance, showed significantly reduced protein levels when analyzed by Western blotting with two different antibodies recognizing different epitopes .

Domain-Specific Analysis:
MIB2 contains several functional domains including RING finger domains (responsible for E3 ligase activity) and ankyrin repeats (important for protein-protein interactions). Variants in these domains may have distinct functional consequences. The V742G variant is positioned in a highly conserved ankyrin repeat (amino acids 696-780), potentially disrupting the hydrophobic packing between α-helices and destabilizing the helix-turn-helix fold .

Structure-Function Correlations:
Clustering of disease-related variants in specific regions of the three-dimensional structure provides supporting evidence for pathogenicity. Known cancer-related variants such as A752V (liver) and D775N (lung) are positioned in the same ankyrin repeat near V742G, suggesting this region is functionally important .

What are the recommended protocols for MIB2 antibody-based detection in Western blotting?

For optimal Western blot detection of MIB2 using biotin-conjugated antibodies:

Sample Preparation:

• Lyse cells in RIPA buffer containing protease inhibitors

• Quantify protein concentration using BCA or Bradford assay

• Load 20-40 μg total protein per lane

• Include positive control (e.g., HeLa cell lysate) and negative control (MIB2 knockdown sample)

Blotting Protocol:

• Separate proteins on 8-10% SDS-PAGE gel (MIB2 MW ~100 kDa)

• Transfer to PVDF membrane (nitrocellulose alternative acceptable)

• Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Incubate with MIB2 Antibody, Biotin conjugated (1:500-1:1000) overnight at 4°C

• Wash 3x with TBST, 10 minutes each

• Incubate with streptavidin-HRP (1:2000-1:5000) for 1 hour at room temperature

• Wash 3x with TBST, 10 minutes each

• Develop using ECL substrate

Signal Amplification:
For low-abundance samples, implement tyramide signal amplification:

• After primary antibody incubation, apply HRP-conjugated streptavidin (1:1000)

• Incubate with Biotin XX tyramide solution for 2-10 minutes

• Wash thoroughly

• Apply streptavidin-HRP again (1:5000)

• Develop with ECL substrate

This approach has been shown to increase detection sensitivity by 10-50 fold compared to standard methods .

What controls should be included when using MIB2 Antibody, Biotin conjugated in experimental setups?

Proper experimental controls are critical for generating reliable data with MIB2 Antibody, Biotin conjugated:

Antibody Specificity Controls:

• Positive Control: Include samples known to express MIB2 (e.g., HeLa cells)

• Negative Control: Include MIB2 knockout/knockdown samples or tissues

• Isotype Control: Include matched isotype IgG, biotin-conjugated, to identify non-specific binding

Biotin-Specific Controls:

• Endogenous Biotin Control: Include a sample processed without primary antibody but with streptavidin conjugate to detect endogenous biotin

• Biotin Blocking Control: For tissues with high endogenous biotin (liver, kidney, brain), include samples treated with a biotin blocking kit

• Avidin/Streptavidin Blocking Control: Include samples with avidin/streptavidin blocking reagent to confirm specificity

Protein Interaction Controls:
When studying MIB2-CYLD interactions, NEMO protein serves as a positive control for CYLD interaction partners, as it is a well-established binding partner of CYLD .

Antibody Dilution Controls:
Include a dilution series of the primary antibody to determine optimal signal-to-noise ratio for your specific application and tissue/cell type.

How can MIB2 expression be quantified in tissue samples using immunohistochemistry?

Quantifying MIB2 expression in tissue samples requires systematic analysis:

Sample Processing:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours

• Process and embed in paraffin

• Section at 4-5 μm thickness

• Deparaffinize and rehydrate sections

• Perform antigen retrieval using high-pressure citrate buffer (pH 6.0)

• Block with 10% normal goat serum for 30 minutes at room temperature

Staining Protocol:

• Incubate with MIB2 antibody (1:500-1:600) overnight at 4°C

• Wash thoroughly with PBS

• Apply biotinylated secondary antibody

• Visualize using an HRP-conjugated streptavidin system

Quantification Methods:

• H-Score Method: Multiply staining intensity (0-3) by percentage of positive cells (0-100%) to generate scores ranging from 0-300

• Digital Image Analysis: Use software platforms (e.g., QuPath, ImageJ) to quantify staining intensity and percentage of positive cells

• Reference Standards: Include control samples with known MIB2 expression levels in each batch

What are common troubleshooting strategies for MIB2 Antibody, Biotin conjugated experiments?

When encountering issues with MIB2 Antibody, Biotin conjugated, consider the following troubleshooting approaches:

High Background Issues:

• Increase blocking time/concentration (try 5% BSA instead of serum)

• Use specific biotin/avidin blocking kits if tissues have high endogenous biotin

• Increase washing steps duration and frequency

• Reduce primary and secondary antibody concentrations

• Use 0.05% Tween-20 in washing buffers to reduce non-specific binding

Weak or No Signal:

• Verify MIB2 expression in your sample (check literature or databases)

• Optimize antigen retrieval methods (try different pH buffers or heat-induced epitope retrieval)

• Increase antibody concentration or incubation time

• Apply signal amplification systems like tyramide signal amplification

• Check antibody storage conditions and avoid freeze-thaw cycles

Protein-Protein Interaction Detection:
When studying MIB2-CYLD interactions, consider that different methodologies may yield complementary information. AlphaScreen assays, GST-pulldown experiments, and co-immunoprecipitation approaches have all been successfully used to detect MIB2-CYLD interactions .

How can researchers investigate MIB2's role in ubiquitination pathways using biotin-conjugated antibodies?

Investigating MIB2's E3 ligase activity requires specialized approaches:

Ubiquitination Assay Protocol:

• Express and purify recombinant wild-type MIB2 and variant forms (e.g., V742G, catalytically inactive mutants)

• Combine with E1, E2, and FLAG-tagged ubiquitin proteins in an in vitro reaction

• Analyze auto-ubiquitination activity via western blotting using anti-FLAG antibodies

• Compare wild-type and variant MIB2 activities quantitatively

Ubiquitin Chain-Type Analysis:
To distinguish between different ubiquitin chain types (critical for determining functional outcomes):

• Use specific antibodies capable of detecting either Lys-48- or Lys-63-linked polyubiquitin

• Alternatively, employ ubiquitin mutants lacking ubiquitination sites except for Lys-48 or Lys-63

• MIB2 has been shown to catalyze Lys-48-linked polyubiquitination of CYLD, but not Lys-63-linked chains

Target Identification:
To identify ubiquitination sites on MIB2 targets:

• Perform LC-MS/MS analysis on K-ε-GG antibody immunoprecipitates

• This approach successfully identified Lys-338 and Lys-530 as MIB2 ubiquitination sites on CYLD

• Confirm findings by generating lysine-to-arginine mutants (K338R, K530R) and testing their resistance to MIB2-mediated degradation

Protein Stability Assessment:
To evaluate MIB2's effect on target protein stability:

• Perform cycloheximide chase experiments comparing wild-type and catalytically inactive MIB2

• Monitor protein half-life of targets (e.g., CYLD) in the presence/absence of MIB2

• Include ubiquitination site mutants as controls (e.g., CYLD-K338/530R has been shown to be stabilized even in the presence of overexpressed MIB2)

How is MIB2 Antibody, Biotin conjugated being used in inflammation and immunity research?

MIB2 Antibody, Biotin conjugated provides valuable insights into inflammatory mechanisms:

NF-κB Signaling Studies:
MIB2 enhances NF-κB activation through CYLD degradation, making it a potential therapeutic target for inflammatory conditions. Biotin-conjugated MIB2 antibodies enable researchers to track MIB2 expression and localization in various inflammatory models and human tissue samples .

Arthritis Models:
The K/BxN serum-transfer arthritis model has demonstrated the importance of MIB2 in inflammatory responses. In this model, Mib2-knockout mice exhibited reduced serum IL-6 levels and suppressed inflammatory responses, highlighting MIB2's pro-inflammatory role .

TNFα-Mediated Signaling:
MIB2 has been shown to enhance NF-κB signaling via TNFα stimulation. Biotin-conjugated MIB2 antibodies allow researchers to investigate the spatiotemporal dynamics of MIB2 in response to TNFα and other inflammatory stimuli .

LUBAC Complex Interactions:
MIB2's relationship with the linear ubiquitination assembly complex (LUBAC) represents an emerging area of research. Biotinylated antibodies facilitate co-localization studies to examine MIB2's spatial relationship with LUBAC components during inflammatory signaling .

What role does MIB2 play in pathological conditions, and how can MIB2 antibodies advance this research?

MIB2 has been implicated in several pathological conditions:

Cardiac Abnormalities:
MIB2 variants have been associated with left ventricle hypertrabeculation/non-compaction, potentially through alterations in NOTCH signaling. MIB2 Antibody, Biotin conjugated can be used to compare expression patterns between normal and affected cardiac tissues .

Ménétrier-like Gastropathy:
MIB2 variants segregate with Ménétrier-like gastropathy in affected families. Immunohistochemical analysis using biotin-conjugated MIB2 antibodies can help characterize MIB2 expression in gastric tissues from affected individuals .

Multiple Familial Trichoepitheliomas:
MIB2 significantly enhances degradation of the missense CYLD P904L variant found in multiple familial trichoepitheliomas. This finding suggests a potential mechanism for how MIB2 might contribute to skin tumor development in this condition .

Cancer Research:
Cancer-related MIB2 variants (e.g., A752V in liver cancer, D775N in lung cancer) cluster in the ankyrin repeat domain near the V742 position associated with other pathologies. This suggests a functional hotspot that could be targeted for therapeutic development .

Future Therapeutic Potential: As E3 ubiquitin ligases become increasingly recognized as druggable targets, characterization of MIB2 expression and activity using biotinylated antibodies may facilitate development of specific inhibitors for inflammatory conditions and cancers where MIB2 is dysregulated.