HIPP47 Antibody
Description
Introduction to HSP47 Antibody
HSP47 antibody is an immunological reagent specifically designed to detect and bind to Heat Shock Protein 47, a critical molecular chaperone involved in collagen biosynthesis. The antibody is a valuable tool in both research and diagnostic applications, enabling scientists and clinicians to investigate HSP47's expression and distribution in various tissues and cell types.
The antibody discussed in the available research is a monoclonal mouse IgG antibody, specifically developed to target human HSP47. This particular antibody (catalog # MAB91662) is developed using Chinese hamster ovary cell line CHO-derived recombinant human HSP47, specifically the amino acid sequence Ala19-Asp412, corresponding to accession number P50454 . The high specificity of this antibody makes it valuable for precise detection of human HSP47 in various experimental settings.
Specificity and Reactivity
One of the key characteristics of the HSP47 antibody (clone #950828) is its high specificity for human HSP47. It effectively detects human HSP47 in direct Enzyme-Linked Immunosorbent Assays (ELISAs) and Western blot applications. Importantly, the antibody shows no cross-reactivity with mouse HSP47 in Western blot applications, highlighting its species-specific nature . This specificity is crucial for research applications where precise targeting of human HSP47 is required without interference from murine proteins.
IgG Subclass Considerations
While the specific subclass of the described HSP47 antibody is not explicitly stated, it's important to understand how IgG subclass affects antibody properties. Research on monoclonal antibodies indicates that IgG subclass significantly influences molecular properties, either through direct effects or via interaction with the variable region. For instance, IgG1 monoclonal antibodies tend to exhibit higher solubility than either IgG2 or IgG4 monoclonal antibodies in common pH 6 buffer matrices . These properties may influence the performance of HSP47 antibodies depending on their specific IgG subclass.
Western Blot Analysis
The HSP47 antibody has demonstrated effective performance in Western blot applications. In experimental settings, it successfully detected HSP47 in lysates from multiple human cell lines, including HeLa (human cervical epithelial carcinoma), HepG2 (human hepatocellular carcinoma), A549 (human lung carcinoma), and JAR (human choriocarcinoma) cell lines. When using the antibody in Western blot protocols, a specific band for HSP47 was detected at approximately 47 kDa, confirming the expected molecular weight of the target protein .
In typical Western blot protocols using this antibody, PVDF membranes were probed with 0.1 μg/mL of the Mouse Anti-Human HSP47 Monoclonal Antibody, followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody. These experiments were conducted under reducing conditions using specific immunoblot buffer groups .
Immunohistochemistry
The HSP47 antibody has proven valuable for immunohistochemistry applications, particularly in paraffin-embedded tissue sections. In research applications, the antibody successfully detected HSP47 in immersion-fixed paraffin-embedded sections of human breast cancer tissue. For optimal results, the antibody was applied at a concentration of 25 μg/mL and incubated overnight at 4°C .
In these immunohistochemistry applications, tissue was typically stained using Anti-Mouse HRP-DAB Cell & Tissue Staining Kits, producing a brown stain where HSP47 was present. Counterstaining with hematoxylin (blue) helps provide cellular context. Notably, the staining pattern revealed that HSP47 was specifically localized to the cytoplasm of cells in breast cancer tissue .
Simple Western Analysis
The HSP47 antibody has also been validated for use in Simple Western applications, a capillary-based immunoassay platform that automates Western blotting procedures. This technique was successfully used to detect HSP47 in lysates of HeLa and HepG2 cell lines, providing an alternative to traditional Western blotting with potentially improved reproducibility and quantification .
HSP47 Expression in Human Cancer Cell Lines
Research using the HSP47 antibody has revealed important patterns of HSP47 expression across various human cancer cell lines. Western blot and Simple Western analyses have demonstrated the presence of HSP47 in multiple cell types, suggesting its widespread expression in diverse cancer lineages. The detection of HSP47 in HeLa, HepG2, A549, and JAR cell lines indicates its expression in cervical, liver, lung, and placental cancer cells, respectively .
This widespread expression across different cancer types suggests that HSP47 may play important roles in various cancer pathophysiologies, potentially related to its function in collagen biosynthesis and processing, which is critical for tumor microenvironment remodeling and metastasis.
HSP47 in Breast Cancer Tissue
Immunohistochemistry studies using the HSP47 antibody have provided valuable insights into the expression pattern of HSP47 in human breast cancer tissue. The cytoplasmic localization of HSP47 staining is consistent with its known function as an endoplasmic reticulum resident chaperone involved in collagen biosynthesis .
The presence of HSP47 in breast cancer tissue may have implications for understanding tumor development, progression, and potential therapeutic approaches. The specific cytoplasmic staining pattern could indicate increased collagen production or processing in breast cancer cells, which might contribute to tumor stroma formation and cancer cell invasion.
Impact of IgG Subclass on Antibody Properties
While not specific to HSP47 antibodies, research on antibody properties provides relevant context for understanding how different IgG subclasses might affect HSP47 antibody performance. Studies have shown that IgG subclass plays a significant role in determining molecular properties of antibodies, either through direct effects or via interaction with the variable region .
Hydrophobicity Across IgG Subclasses
| IgG subclass | Fv A (RT, min) | Fv B (RT, min) | Fv C (RT, min) | Fv A (HIP%) | Fv B (HIP%) | Fv C (HIP%) |
|---|---|---|---|---|---|---|
| IgG1 | 13.4 | 13.0 | 16.0 | 42.1 | 40.1 | 55.0 |
| IgG1EN | 13.4 | 13.0 | 16.0 | 42.1 | 40.0 | 55.2 |
| IgG2 | 13.5 | 13.0 | 16.1 | 42.3 | 40.1 | 55.7 |
| IgG4PAA | 14.0 | 13.6 | 16.4 | 44.8 | 43.0 | 56.9 |
This data demonstrates that while the variable region primarily determines hydrophobicity, subtle differences exist between IgG subclasses that may affect antibody performance in specific applications .
Immunoassay Principles
Understanding the principles of immunoassays is important for effective use of HSP47 antibodies. Immunoassays measure the presence of specific antibodies using various antibody-capturing platforms such as ELISA-based methods, particle gel techniques, or turbidimetry. These assays typically offer high sensitivity (>99%), making them ideal as initial screening tests .
Relevance to HSP47 Antibody Applications
While the search results don't specifically address HSP47 antibody-based immunoassays, the general principles of immunoassay development and validation apply to HSP47 detection systems. When developing or selecting HSP47 antibody-based assays, consideration should be given to the balance between sensitivity and specificity, as well as the appropriate thresholds for positive results based on the intended application.
Humanized Mouse Models for Antibody Testing
Although not directly related to HSP47 antibodies, advances in antibody testing platforms provide important context for future development of HSP47 antibody applications. Recent research has developed humanized mouse strains in which human extracellular domains (ECD) of proteins replace their murine counterparts using CRISPR/Cas9 gene-editing .
Such humanized models could potentially be valuable for testing HSP47 antibodies in vivo, particularly if there are significant differences between human and mouse HSP47 proteins. The described HSP47 antibody shows no cross-reactivity with mouse HSP47, highlighting the need for humanized models to study human HSP47 function in vivo .
Product Specs
Composition: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
KEGG: ath:AT3G20180
UniGene: At.63564
Q&A
What validation methods should be used to confirm HIPP47 antibody specificity?
When validating HIPP47 antibody specificity, genetic approaches have proven more reliable than orthogonal strategies. Data shows that antibodies validated through genetic strategies (using knockout or knockdown controls) have significantly higher confirmation rates (80%) compared to orthogonal methods, particularly for immunofluorescence applications . For Western blot validation, both genetic and orthogonal approaches can be effective, with success rates of 89% and 80% respectively . Always include knockout controls when possible to establish definitive specificity profiles.
How does HIPP47 antibody perform across different experimental applications?
Performance varies by application and antibody format. Based on comprehensive antibody validation studies, expect differential performance across Western blot (WB), immunoprecipitation (IP), and immunofluorescence (IF) applications. Research indicates that recombinant antibodies generally outperform monoclonal and polyclonal variants across applications:
| Antibody Type | Western Blot | Immunoprecipitation | Immunofluorescence |
|---|---|---|---|
| Recombinant | 67% | 54% | 48% |
| Monoclonal | 41% | 32% | 31% |
| Polyclonal | 27% | 39% | 22% |
Success rates by antibody type and application
What essential controls should be included when using HIPP47 antibody?
Always include genetic controls when validating HIPP47 antibody. Research demonstrates that knockout (KO) cell lines provide the most definitive control for antibody validation . For Western blot experiments, include both wildtype and KO cell lysates to verify band specificity. For immunofluorescence, parallel staining of wildtype and KO cells is essential, as approximately 22% of published IF studies used antibodies that failed specificity validation . Additional controls should include secondary-only controls and isotype controls to account for non-specific binding.
How can computational modeling enhance HIPP47 antibody design for custom specificity?
Computational modeling can significantly improve HIPP47 antibody specificity through biophysics-informed approaches. Recent research demonstrates that models trained on experimentally selected antibodies can identify distinct binding modes associated with specific ligands . This approach enables:
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Prediction of antibody performance against new ligand combinations
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Generation of novel antibody sequences with predefined binding profiles
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Design of antibodies with either cross-specific binding (interacting with multiple ligands) or highly specific binding (targeting a single ligand while excluding others)
The optimization process involves minimizing energy functions (Esw) for desired ligand interactions while maximizing energy functions for undesired ligands .
What methodological approaches can disentangle HIPP47 binding modes against similar targets?
When HIPP47 must discriminate between structurally similar targets, implement a methodology that integrates experimental selection with computational analysis. This approach has successfully identified different binding modes even for chemically similar ligands . Key steps include:
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Conduct phage display selections against various combinations of target ligands
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Build a computational model expressing selection probability (p) in terms of selected and unselected binding modes
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Implement a mathematical framework where each mode (w) is described by experiment-dependent (μwt) and sequence-dependent (Ews) parameters
This approach enables identification of antibody sequences that bind specifically to desired targets while avoiding cross-reactivity with similar epitopes.
How does the recombinant format of HIPP47 compare to monoclonal or polyclonal versions?
Recombinant antibodies consistently outperform other formats across applications. Comprehensive analysis of 614 commercially available antibodies shows recombinant antibodies demonstrate superior performance in all testing conditions . For HIPP47 specifically, expect the recombinant format to provide:
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Significantly higher success rates in Western blot applications (67% vs. 41% for monoclonal and 27% for polyclonal)
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Better performance in immunoprecipitation (54% vs. 32-39% for other formats)
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Superior reliability in immunofluorescence (48% vs. 31% for monoclonal and 22% for polyclonal)
Additionally, recombinant antibodies offer better reproducibility and reduced batch-to-batch variation, which is particularly important for longitudinal studies.
What mechanisms should be considered when using HIPP47 for therapeutic applications?
If HIPP47 targets CD47 pathways, consider the mechanisms of phagocytosis regulation by innate immune cells. CD47 functions as a regulator of phagocytosis mediated by macrophages and dendritic cells, serving as a ligand for SIRP-alpha receptor . Research demonstrates increased expression of CD47 on primary human acute myeloid leukemia (AML) stem cells, with blocking antibodies enabling phagocytosis and elimination of AML . When designing therapeutic applications:
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Evaluate HIPP47's ability to block CD47-SIRP-alpha interaction
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Assess potential to enable phagocytosis of target cells
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Consider combination therapies that might enhance therapeutic efficacy
How can non-specific binding be minimized when working with HIPP47 antibody?
Non-specific binding can significantly impact experimental outcomes. To minimize this issue:
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Implement extensive pre-adsorption protocols against potential cross-reactive targets
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Utilize computational modeling to predict and mitigate cross-reactivity based on binding energy functions
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For immunoprecipitation experiments, include appropriate pre-clearing steps with control beads
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Consider sequential purification strategies that can enhance specificity
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Optimize blocking conditions for each specific application to minimize background
Research indicates that for Western blot applications, approximately 9/65 antibodies demonstrated non-specific binding to unrelated proteins that wasn't eliminated in knockout controls .
What are the implications of published research using unvalidated antibodies?
The scientific impact of unvalidated antibodies is substantial. Bibliometric analysis reveals:
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31% of Western blot publications used antibodies that failed specificity validation
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35% of immunoprecipitation studies used antibodies unable to capture target proteins
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22% of immunofluorescence publications employed antibodies that couldn't localize their targets
This indicates that 20-30% of published figures may be generated using antibodies that don't recognize their intended targets, highlighting the critical importance of thorough validation before using HIPP47 or any research antibody .
What parameters should be optimized when HIPP47 produces inconsistent results?
When troubleshooting inconsistent HIPP47 performance:
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Antibody concentration: Titrate across a wide range to determine optimal signal-to-noise ratio
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Incubation conditions: Systematically vary temperature, time, and buffer composition
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Sample preparation: Evaluate different lysis methods, fixation protocols, and antigen retrieval techniques
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Detection systems: Compare direct labeling vs. secondary antibody detection
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Blocking reagents: Test various blocking agents (BSA, milk, commercial blockers) for reduced background
Document all optimization steps methodically to ensure reproducibility across experiments and research teams.
How can HIPP47 be integrated into multi-parameter experimental designs?
For complex experimental designs requiring multiple antibodies:
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Verify antibody compatibility in multiplexed protocols through sequential staining validation
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Establish spectral compensation matrices when using fluorescently-labeled antibodies
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Determine optimal antibody pairs for co-immunoprecipitation studies
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Validate epitope accessibility in various experimental conditions
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Consider antibody conjugation strategies that minimize steric hindrance
When designing experiments, remember that approximately 40% of proteins lack successful antibodies for immunofluorescence applications, which may necessitate alternative detection strategies .
