DAB2IP Antibody, HRP conjugated

What is DAB2IP and why is it an important research target?

DAB2IP (DOC-2/DAB2 interactive protein) is a tumor suppressor belonging to the RAS-GTPase-activating protein (RAS-GAP) family. It plays crucial roles in regulating cell survival, apoptosis, and epithelial-to-mesenchymal transition through inhibiting several pathways . DAB2IP is frequently downregulated in multiple cancers including renal cell carcinoma and colorectal cancer, making it a significant target for cancer research . Mechanistically, DAB2IP can inhibit p53 ubiquitin-mediated degradation by competitively binding to GRP75, thereby stabilizing p53 protein levels and promoting tumor-suppressive characteristics .

What does HRP conjugation mean for DAB2IP antibodies?

HRP (Horseradish Peroxidase) conjugation refers to the chemical attachment of the enzyme to an antibody molecule, creating a direct detection system. For DAB2IP antibodies, HRP conjugation eliminates the need for secondary antibody incubation, simplifying experimental workflows and potentially reducing background signals . The HRP enzyme catalyzes a chemiluminescent reaction when exposed to appropriate substrates, enabling direct visualization of DAB2IP in applications like Western blotting, immunohistochemistry, and ELISA.

How do unconjugated DAB2IP antibodies compare with HRP-conjugated versions?

Unconjugated DAB2IP antibodies (such as 23582-1-AP) utilize a two-step detection system requiring primary and secondary antibody incubations, which provides signal amplification but increases experiment time . In contrast, HRP-conjugated antibodies offer direct detection but with approximately 30x lower signal intensity due to less specific activity and removal of secondary antibody amplification . This signal reduction can be advantageous when detecting abundantly expressed proteins to prevent saturation, but may challenge detection of low DAB2IP expression in certain cancer models where the protein is downregulated.

What are the optimal dilution ratios for DAB2IP antibodies in different applications?

For unconjugated DAB2IP antibodies (like 23582-1-AP), the recommended dilutions are:

• Western Blot: 1:2000-1:16000

• Immunoprecipitation: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Immunofluorescence and Immunohistochemistry: Sample-dependent, requiring optimization

For HRP-conjugated antibodies, dilutions typically need adjustment compared to unconjugated versions. Based on HRP-conjugated antibody principles, starting dilutions around 1:50 to 1:100 are appropriate with optimization required for each specific application . The optimal dilution should provide specific signal without background or saturation issues.

How can DAB2IP HRP-conjugated antibodies be effectively used in multiplexing experiments?

Multiplexing with DAB2IP HRP-conjugated antibodies requires careful signal balancing between different target proteins. When multiplexing with proteins that produce stronger signals (like housekeeping proteins), direct detection with HRP-conjugated antibodies helps bring chemiluminescent signals to comparable levels . For example:

When multiplexing DAB2IP detection with proteins like GAPDH, using HRP-conjugated anti-GAPDH can bring signals closer together for optimal chemiluminescent detection within linear ranges .

What sample preparation methods optimize DAB2IP detection?

For optimal DAB2IP antibody detection, sample preparation should include:

• Cell lysis using buffers containing protease inhibitors to prevent degradation

• Proper denaturation for Western blotting by heating at 95°C for 5 minutes in sample buffer

• For immunoprecipitation, using 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate

• When working with tissue samples, thorough homogenization followed by membrane protein extraction is critical, as demonstrated in studies with mouse and rat brain tissue

How can signal saturation issues be addressed when using DAB2IP HRP-conjugated antibodies?

Signal saturation is a common challenge in protein detection studies. For DAB2IP HRP-conjugated antibodies, several approaches can effectively manage this issue:

• Antibody dilution adjustment: Create a dilution series to identify the minimal concentration providing sufficient specific signal

• Mixing labeled and unlabeled antibodies: Adding unlabeled antibodies while maintaining total antibody concentration can reduce signal without affecting specificity

• Secondary antibody optimization: When using a two-step system, mixing different ratios of unlabeled:HRP-labeled secondary (from 5:1 up to 640:1) allows signal fine-tuning

• Direct HRP-conjugation: Substituting a primary antibody with its HRP-conjugated counterpart reduces signal approximately 30-fold compared to indirect detection methods

What are effective strategies for validating DAB2IP antibody specificity?

Validating DAB2IP antibody specificity requires multiple approaches:

• Testing in cells with verified DAB2IP expression (A431, HeLa, HepG2 cells, and brain tissue)

• Confirming the molecular weight of detected bands aligns with expected DAB2IP size (approximately 118 kDa observed, though calculated MW is 132 kDa)

• Using RNA interference to knock down DAB2IP expression and observe corresponding signal reduction

• For HRP-conjugated antibodies, comparing detection patterns with unconjugated versions of the same antibody clone

• Implementing peptide competition assays where pre-incubation with immunizing peptide should abolish specific binding

How can researchers interpret complex banding patterns in DAB2IP Western blots?

When analyzing DAB2IP Western blots, researchers may observe multiple bands due to:

• Post-translational modifications affecting protein migration

• Alternative splicing variants of DAB2IP

• Proteolytic cleavage products

The primary band expected for full-length DAB2IP is approximately 118 kDa, which differs slightly from the calculated molecular weight of 132 kDa (based on 1189 amino acids) . Validation across multiple cell lines confirms this pattern. When using HRP-conjugated antibodies, band intensities may appear lower than with indirect detection systems, while maintaining the same molecular weight profile.

How can DAB2IP HRP-conjugated antibodies be utilized to study protein-protein interactions?

DAB2IP HRP-conjugated antibodies offer unique advantages for studying protein-protein interactions:

• For co-immunoprecipitation experiments exploring DAB2IP interactions with proteins like GRP75, direct detection with HRP-conjugated antibodies eliminates potential cross-reactivity from secondary antibodies

• Research has demonstrated that DAB2IP does not directly interact with p53 but rather competes with p53 for binding to GRP75, preventing p53 ubiquitination and degradation

• Immunoprecipitation-mass spectrometry (IP-MS) identified 166 proteins interacting with both DAB2IP and p53, with GRP75, HSP90AA1, and other chaperones classified in the "ubiquitin protein ligase binding" category

The competitive binding mechanism between DAB2IP and p53 for GRP75 represents a novel regulatory mechanism that could be further explored using HRP-conjugated antibodies in proximity-dependent detection systems.

What role does DAB2IP play in radiation response of cancer cells?

DAB2IP plays a critical role in radiation sensitivity of cancer cells:

• DAB2IP-deficient renal cell carcinoma cells acquire resistance to ionizing radiation (IR)

• Mechanistically, DAB2IP can form a complex with PARP-1 and E3 ligases responsible for degrading PARP-1, with elevated PARP-1 levels associated with IR resistance in RCC cells

• DAB2IP can directly interact with PARP-1 protein and affect its turnover by recruiting E3-ligases (RanBP2, TRIP12, and RNF40)

• PARP-1 inhibitors can enhance the IR response in both RCC xenograft models and patient-derived xenograft (PDX) models with DAB2IP deficiency

These findings suggest DAB2IP HRP-conjugated antibodies could be valuable tools for studying radiation response mechanisms in various cancer models.

How does DAB2IP regulate p53 stability and what methods can detect this interaction?

DAB2IP regulates p53 stability through a competitive binding mechanism:

• DAB2IP inhibits ubiquitin-proteasome-dependent p53 degradation without directly interacting with p53

• DAB2IP competes with p53 for binding to GRP75, thereby preventing GRP75-mediated p53 ubiquitination and degradation

• With declining DAB2IP protein levels, GRP75 binding capacity to p53 gradually increases, leading to enhanced p53 degradation

• This regulatory mechanism represents a novel component of CHIP-mediated p53 degradation

To detect this interaction network, researchers can employ immunoprecipitation followed by Western blotting with HRP-conjugated antibodies for direct visualization of the competitive binding dynamics between DAB2IP, GRP75, and p53.

What controls should be included when using DAB2IP HRP-conjugated antibodies?

Proper experimental controls are essential when working with DAB2IP HRP-conjugated antibodies:

• Positive controls: Cell lines with verified DAB2IP expression (A431, HeLa, HepG2 cells, or brain tissue)

• Negative controls: Samples with DAB2IP knockdown via siRNA or from DAB2IP-knockout models

• Technical controls: Omission of primary antibody to assess non-specific binding

• Loading controls: When multiplexing with housekeeping proteins, using directly conjugated anti-GAPDH-HRP can help balance signal intensities

• Antibody validation controls: Comparison between HRP-conjugated and unconjugated versions of the same antibody clone

How should researchers quantify DAB2IP expression in multiplex experiments?

Accurate quantification of DAB2IP expression in multiplex experiments requires careful methodological consideration:

• Capture images within the linear dynamic range of detection, avoiding saturated signals

• For multiplexing with housekeeping proteins, consider the significantly different signal intensities between direct and indirect detection (approximately 30-fold difference)

• When signal intensities differ dramatically between DAB2IP and reference proteins, adjust antibody concentrations or use direct HRP-conjugated versions of reference antibodies to balance signal strengths

• For Western blot quantification, the chemiluminescent signal from directly detected proteins using HRP-conjugated antibodies is significantly lower than indirectly detected signals, requiring appropriate exposure time optimization

What are the key considerations when optimizing exposure times for DAB2IP detection?

Optimizing exposure times is critical for accurate DAB2IP detection and quantification:

• For HRP-conjugated antibodies, shorter exposure times may be required for abundant proteins to prevent saturation

• When multiplexing different targets with varying expression levels, finding a common exposure time can be challenging

• A practical approach involves using HRP-conjugated antibodies for highly expressed proteins (like GAPDH) while using traditional indirect detection for lower-abundance targets

• When comparing indirect detection (using 1:50 mouse anti-GAPDH and anti-mouse HRP secondary) versus direct detection (using 1:50 anti-GAPDH-HRP), the directly detected signal produces approximately 30-fold lower chemiluminescence

This balanced approach ensures optimal signal detection within the linear range for accurate quantification of both DAB2IP and reference proteins.