ID4 Antibody

What is ID4 protein and why is it important in research?

ID4 (Inhibitor of DNA binding 4) is a dominant negative helix-loop-helix protein that negatively regulates basic helix-loop-helix (bHLH) transcription factors by forming heterodimers and inhibiting their DNA binding and transcriptional activity . It lacks a basic DNA binding domain while retaining the ability to form protein-protein interactions. ID4 is critically involved in the regulation of many cellular processes during both prenatal development and tumorigenesis, making it an important target for fundamental and translational research . Recent studies have established that ID4 levels dictate the stem cell state in mouse spermatogonia, highlighting its importance in stem cell biology .

What species reactivity can be expected from commercially available ID4 antibodies?

Most commercially available ID4 antibodies show confirmed reactivity with human, mouse, and rat samples . Some antibodies, like the rabbit polyclonal antibody described in search result , may have predicted reactivity with additional species such as pig and dog, though these predictions are based primarily on sequence homology and should be experimentally validated. When selecting an ID4 antibody for non-standard model organisms, researchers should carefully check the manufacturer's validation data or conduct their own validation studies to confirm cross-reactivity.

What are the optimal dilutions for using ID4 antibodies in different applications?

For Western blot (WB) applications, the recommended dilution range for ID4 antibody (21803-1-AP) is 1:1000-1:4000 . For immunohistochemistry (IHC), the appropriate dilution range is 1:50-1:500 . It's essential to note that the optimal dilution should be determined empirically for each specific experimental setup and sample type. As stated in the technical information, "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" . A titration experiment with a range of antibody dilutions is advisable when first establishing a new protocol or when working with a new sample type.

What antigen retrieval methods are recommended for using ID4 antibodies in immunohistochemistry?

For optimal immunohistochemical detection of ID4, antigen retrieval with TE buffer at pH 9.0 is suggested . Alternatively, antigen retrieval may be performed using citrate buffer at pH 6.0, although this appears to be a secondary recommendation . The choice between these methods may depend on tissue fixation conditions, tissue type, and the specific ID4 epitope targeted by the antibody. Researchers should compare both methods when establishing IHC protocols for ID4 detection to determine which provides the optimal signal-to-noise ratio for their specific samples.

How should ID4 antibodies be stored to maintain optimal activity?

ID4 antibodies should be stored at -20°C, where they remain stable for one year after shipment . The storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Importantly, aliquoting is generally unnecessary for -20°C storage of these antibodies, though this may vary between manufacturers. Some preparations, particularly smaller volume sizes (20μl), may contain 0.1% BSA as a stabilizer . When handling the antibody, minimize freeze-thaw cycles and avoid prolonged exposure to room temperature to preserve antibody integrity and specificity.

How can I validate the specificity of an ID4 antibody for my experimental system?

Validating ID4 antibody specificity requires multiple approaches. First, include positive controls using cell lines known to express ID4, such as HepG2, K-562, or MDA-MB-231 cells, and tissue samples like mouse or rat testis . Second, employ negative controls using ID4 knockout or knockdown systems. Third, conduct Western blot analysis to confirm detection at the expected molecular weight range (25-30 kDa) . For immunohistochemistry validation, compare staining patterns with published literature and confirm localization consistent with ID4's transcriptional regulatory function. Finally, performing antibody validation using multiple detection methods (WB, IHC, IF) provides stronger evidence of specificity than relying on a single technique.

What cell lines and tissue samples are appropriate as positive controls for ID4 antibody experiments?

For Western blot applications, HepG2 cells, K-562 cells, and MDA-MB-231 cells have been validated as positive controls for ID4 detection . For tissue samples, mouse testis tissue has been confirmed as a reliable positive control for both Western blot and immunohistochemistry applications . Rat testis tissue has also been validated for immunohistochemical detection of ID4 . These validated positive controls should be included when establishing new ID4 antibody protocols or when troubleshooting existing ones to ensure proper antibody function and specificity.

What are the recommended techniques for co-immunoprecipitation studies involving ID4?

For co-immunoprecipitation (co-IP) of ID4 and its interaction partners, the rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) technique has proven effective in both cell lines and patient-derived xenograft models . When designing co-IP experiments, use biological triplicates (each with technical duplicates or triplicates) followed by mass spectrometry to ensure reliability. Include appropriate IgG controls to identify non-specific binding. For validation of specific interactions, such as the ID4-MDC1 interaction, quantitative mass spectrometry methods like SWATH (Sequential Window Acquisition of all THeoretical mass spectra) can provide additional confidence in the results . When analyzing potential interaction partners, consider using Gene Set Enrichment Analysis (GSEA) to identify functionally related groups of proteins.

How is ID4 involved in DNA damage response pathways and what methodologies can detect these interactions?

ID4 interacts with DNA damage response proteins, particularly MDC1 (mediator of DNA damage checkpoint 1), as demonstrated through proteomics approaches . This interaction has been validated in multiple cell lines (HCC70, MDA-MB-468, HCC1954, OVKATE) and in patient-derived xenograft models of triple-negative breast cancer . To study these interactions, researchers can utilize RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins) followed by validation with SWATH (Sequential Window Acquisition of all THeoretical mass spectra) mass spectrometry . ChIP-seq data complements these findings, showing that ID4 interacts with chromatin and nuclear machinery despite lacking a known DNA interaction domain . For researchers investigating ID4's role in DNA damage response, these proteomics approaches provide valuable tools for discovering novel interaction partners.

What is the relationship between ID4 and BRCA1 in breast cancer research?

Proteogenomic analysis has revealed a significant relationship between ID4 and BRCA1 in breast cancer. The BRCA1-PCC (Pearson correlation coefficient) network was the most significantly enriched gene set in the ID4 purified proteome, with 101 out of 852 ID4-associated genes present in the set of 1652 BRCA1-associated genes . This relationship appears particularly relevant in basal-like breast cancer (BLBC), where ID4 is amplified and overexpressed at a higher frequency in BRCA1-mutant BLBC compared with sporadic BLBC . Researchers investigating this relationship should consider integrated proteogenomic approaches, combining ID4 RIME with ChIP-seq and expression analysis to fully characterize the functional interactions between these proteins in different breast cancer subtypes.

How can I design experiments to study ID4's role in stem cell biology?

To investigate ID4's role in stem cell biology, particularly given its established role in dictating stem cell state in mouse spermatogonia , researchers should consider multi-faceted experimental approaches. Begin with expression profiling of ID4 during different stages of stem cell differentiation using validated antibodies for Western blot and immunohistochemistry. For functional studies, implement CRISPR-Cas9 gene editing to create ID4 knockout or knockin models in relevant stem cell lines. Complement this with inducible expression systems to control ID4 levels temporally. Single-cell RNA sequencing before and after ID4 modulation can reveal downstream transcriptional networks. For mechanistic insights, combine ChIP-seq of ID4-interacting partners with ID4 immunoprecipitation studies to identify key protein-protein interactions in the stem cell context. Patient-derived xenograft models can provide translational relevance for findings in cancer stem cell biology.

Why might I observe multiple bands when using ID4 antibodies in Western blot applications?

Multiple bands in ID4 Western blots can occur for several reasons. First, the discrepancy between calculated (17 kDa) and observed (25-30 kDa) molecular weights of ID4 may lead to uncertainty about band identification . Second, post-translational modifications like phosphorylation at S5 and methylation at R49 can create heterogeneity in protein migration . Third, proteolytic degradation of ID4 may generate fragments that are detected by the antibody. Fourth, the antibody might cross-react with other ID family members (ID1, ID2, ID3) due to sequence homology. To address these issues, include positive controls with known ID4 expression, use freshly prepared samples with protease inhibitors, optimize primary antibody concentration, and consider using ID4 knockout/knockdown samples as negative controls to confirm band specificity.

What are the critical factors affecting immunohistochemical detection of ID4 in tissue samples?

Several critical factors affect immunohistochemical detection of ID4. First, tissue fixation conditions significantly impact epitope accessibility; excessive fixation can mask epitopes. Second, the choice of antigen retrieval method is crucial, with TE buffer (pH 9.0) recommended as the primary method for ID4 detection, though citrate buffer (pH 6.0) is an acceptable alternative . Third, antibody dilution must be optimized, with recommended ranges of 1:50-1:500 for IHC applications . Fourth, the specific tissue type matters, with mouse and rat testis tissues serving as validated positive controls . Fifth, blocking conditions must be optimized to reduce background while preserving specific staining. Finally, detection systems (chromogenic vs. fluorescent) should be selected based on the specific research question and the need for co-localization studies with other markers.

How can conflicting results between different ID4 antibodies be reconciled in research studies?

When facing conflicting results between different ID4 antibodies, a systematic reconciliation approach is necessary. First, compare the immunogens used to generate each antibody, as differences in epitope recognition may explain discrepancies. Second, conduct side-by-side validation using known positive (HepG2, K-562 cells) and negative controls (knockdown/knockout samples) . Third, employ multiple detection methods (WB, IHC, IF) to build a consensus view of ID4 expression patterns. Fourth, validate findings with orthogonal techniques not relying on antibodies, such as mRNA expression analysis or CRISPR-based tagging of endogenous ID4. Fifth, consider the possibility that different antibodies may preferentially recognize different post-translationally modified forms of ID4. Finally, when publishing, clearly report which antibody was used (including catalog numbers and lots) and provide all validation data to facilitate comparison across studies.

How can ChIP-seq be optimized for studying ID4 interactions with chromatin?

Optimizing ChIP-seq for ID4 presents unique challenges because ID4 lacks a direct DNA binding domain and instead interacts with chromatin through protein-protein interactions . For successful ChIP-seq experiments, use validated antibodies with confirmed specificity for immunoprecipitation applications. Employ crosslinking conditions that capture both direct and indirect DNA-protein interactions, potentially using dual crosslinking with both formaldehyde and protein-protein crosslinkers like DSG (disuccinimidyl glutarate). Include appropriate controls, such as IgG ChIP and input samples. For data analysis, focus on identifying enrichment patterns that may reflect indirect binding through partner proteins, particularly basic helix-loop-helix transcription factors known to interact with ID4 . Integration with RIME or other proteomics approaches can help identify the protein complexes mediating ID4's interaction with specific genomic regions.

What approaches are recommended for studying ID4's role in tumorigenesis across different cancer types?

To study ID4's role in tumorigenesis across cancer types, implement a comprehensive approach combining multiple methodologies. Begin with expression analysis of ID4 across cancer types using tissue microarrays with validated antibodies . Correlate expression with clinical outcomes using publicly available databases like TCGA. For mechanistic studies, establish cell line models with modulated ID4 expression (overexpression, knockdown, knockout) in relevant cancer types. Assess the effects on cancer hallmarks including proliferation, apoptosis resistance, migration, and invasion. Investigate ID4's interaction with known oncogenic pathways, particularly focused on the BRCA1 network in breast cancer . For in vivo validation, use patient-derived xenograft models that express ID4, similar to the HCI001, HCI002, and HCI009 models described in breast cancer research . Finally, explore the potential of ID4 as a therapeutic target or biomarker through correlation with treatment responses in patient cohorts.

How can researchers effectively combine proteomics and genomics approaches to study ID4 function?

Effective integration of proteomics and genomics for ID4 research requires careful experimental design and data analysis strategies. Begin with ChIP-seq to identify genomic regions associated with ID4 complexes, recognizing that these interactions are likely indirect given ID4's lack of a DNA binding domain . In parallel, conduct RIME to identify protein-protein interactions of ID4, as successfully implemented in cancer cell lines and patient-derived xenografts . Validate key interactions using co-immunoprecipitation followed by Western blot. Perform RNA-seq after ID4 modulation to identify genes regulated directly or indirectly by ID4. For data integration, use computational approaches to correlate ChIP-seq peaks, RIME-identified protein partners, and RNA-seq differentially expressed genes. Pathway and network analyses can reveal functional clusters of ID4-regulated genes and processes. Finally, validate key findings using targeted approaches such as reporter assays for specific genomic regions or CRISPR interference/activation to modulate ID4 target genes.