HMMR Antibody, HRP conjugated

What is HMMR and what cellular functions does it regulate?

HMMR (Hyaluronan mediated motility receptor), also known as CD168, RHAMM, or IHABP, is a multifunctional protein primarily involved in cell motility. When hyaluronan binds to HMMR, it triggers the phosphorylation of several proteins, notably PTK2/FAK1 . HMMR plays crucial roles in cellular transformation and metastasis formation and regulates extracellular-regulated kinase (ERK) activity . Recent studies have demonstrated that HMMR is overexpressed in hepatocellular carcinoma compared to normal tissue and is associated with "G2M checkpoint" and "E2F targets" in RNA sequencing, confirming its role in cell cycle regulation .

What are the applications of HMMR antibody in cancer research?

HMMR antibodies are valuable tools in cancer research, particularly for investigating hepatocellular carcinoma where HMMR is frequently overexpressed . These antibodies can be used for protein detection in various assays, including western blot, immunohistochemistry, and ELISA . They are particularly useful for studying HMMR's role in tumor cell proliferation, migration, and invasion, as HMMR knockdown has been shown to inhibit these processes in HCC cell lines . HMMR antibodies also enable investigation of HMMR's interactions with immune cells and its impact on the tumor microenvironment, as HMMR has been demonstrated to regulate immune cell infiltration and intercellular interactions .

How does the HRP conjugation affect HMMR antibody functionality?

HRP (horseradish peroxidase) conjugation to HMMR antibodies provides a direct enzymatic detection method that eliminates the need for secondary antibodies in immunoassays. The HRP conjugation maintains the specificity of the primary HMMR antibody while enabling colorimetric, chemiluminescent, or fluorescent detection depending on the substrate used . This conjugation is particularly beneficial for ELISA applications, as indicated in the product information . The enzymatic activity of HRP remains stable when stored properly in 50% glycerol with 0.03% Proclin 300 preservative at recommended temperatures, ensuring consistent detection sensitivity over the antibody's shelf life .

What are the recommended storage conditions for HMMR antibody, HRP conjugated?

For optimal preservation of both antibody specificity and HRP enzymatic activity, HMMR antibody-HRP conjugates should be stored in a preservative solution containing 0.03% Proclin 300 and 50% Glycerol in 0.01M PBS at pH 7.4 . These conditions maintain the structural integrity of both the antibody and the conjugated enzyme. While specific temperature recommendations weren't provided in the search results, typical HRP-conjugated antibodies are stored at -20°C for long-term storage with aliquoting recommended to avoid repeated freeze-thaw cycles that can degrade both antibody binding capacity and enzymatic activity of the HRP conjugate.

How can HMMR antibodies be used to investigate immune evasion mechanisms in hepatocellular carcinoma?

Recent studies have uncovered that HMMR facilitates antiphagocytic efficiency in liver cancer cells via the HMMR-CD47 axis . Researchers can utilize HMMR antibodies to investigate this immune evasion mechanism through a multi-faceted approach. Co-immunoprecipitation assays with HMMR antibodies can reveal protein-protein interactions, particularly with CD47, FAK, and SRC, which form signaling complexes in the cytoplasm to activate NF-κB signaling . Immunofluorescence microscopy with HMMR antibodies demonstrates the colocalization of HMMR with CD44 on the cell membrane and with FAK in the cytoplasm . Western blot analysis following HMMR knockdown experiments can confirm the downstream effects on CD47 expression and FAK/SRC phosphorylation status, elucidating the regulatory pathway of the "don't eat me" signal in cancer cells .

What insights can HMMR expression analysis provide for immunotherapy response prediction?

HMMR expression analysis offers significant potential for predicting immunotherapy responses, particularly for immune checkpoint inhibitors targeting the PD-1/PD-L1 pathway. Studies have shown that patients with low HMMR expression might respond more effectively to anti-PD-1 treatment . The mechanistic basis lies in HMMR's regulation of the tumor immune microenvironment, where HMMR knockout has been demonstrated to enhance CD8+ T cell infiltration . Furthermore, HMMR expression positively correlates with indicators of genomic heterogeneity that influence immunotherapy response, including tumor mutational burden (TMB), aneuploidy, homologous recombination deficiency (HRD), and cancer testis antigen (CTA) expression . These correlations provide a rational basis for using HMMR expression as a biomarker for stratifying patients who may benefit from immunotherapy approaches.

How does HMMR contribute to cell cycle regulation and proliferation in cancer cells?

HMMR plays a critical role in cell cycle regulation, particularly in cancer contexts. Through bulk RNA sequencing and single-cell RNA sequencing analyses, HMMR expression has been strongly associated with "G2M checkpoint" and "E2F targets" pathways . Flow cytometry (FCM) confirmation studies have demonstrated HMMR's direct regulatory impact on the cell cycle . At the molecular level, HMMR knockdown experiments using siRNAs have consistently shown inhibition of cancer cell proliferation, as evidenced by colony formation assays and EdU incorporation assays . The proliferation inhibition appears to be mediated through HMMR's interaction with multiple signaling pathways, including the FAK/SRC axis which activates downstream NF-κB signaling . These investigations highlight how HMMR contributes to the dysregulated cell cycle control that is characteristic of cancer progression.

What is the relationship between HMMR and CD47 in tumor immune evasion?

HMMR and CD47 participate in a coordinated immune evasion mechanism in cancer cells. Recent research has revealed that HMMR sustains CD47 expression through a signaling pathway involving FAK/SRC and NF-κB activation . This mechanism enables cancer cells to escape phagocytosis by presenting the "don't eat me" signal (CD47) to macrophages. Clinical data supports this relationship, showing that patients with high expression of both HMMR and CD47 have significantly worse prognosis compared to those with low expression of both markers . Interestingly, HMMR can regulate CD47 expression through two distinct mechanisms: a CD44-dependent pathway where HMMR, CD44, FAK, and SRC form a complex, and a CD44-independent pathway where HMMR directly interacts with FAK and SRC in the cytoplasm . These findings establish HMMR as an upstream regulator of CD47-mediated immune evasion in cancer.

What are the optimal experimental conditions for HMMR knockdown experiments?

For HMMR knockdown experiments, researchers have successfully employed RNA interference techniques using siRNAs targeting specific HMMR sequences. Based on published research, siRNA sequences such as 5'-GCCAACTCAAATCGGAAGTAT-3' and 5'-TCACTTGGTCCTACCTATTAT-3' have demonstrated effective HMMR knockdown . For transfection, Lipofectamine 2000 has been utilized following the manufacturer's protocols . When evaluating knockdown efficiency, western blot analysis using HMMR antibodies (such as Abcam ab124729, diluted 1:1000) is recommended . Multiple siRNAs should be tested initially, with the most effective sequence (e.g., siRNA#3 in the referenced study) selected for subsequent functional assays . For stable knockdown, lentiviral shRNA systems can be employed, with viral production in HEK293T cells and selection of transduced cells using appropriate antibiotics .

How should researchers design co-immunoprecipitation experiments to study HMMR protein interactions?

To effectively study HMMR protein interactions through co-immunoprecipitation (co-IP), researchers should begin by preparing cell lysates under non-denaturing conditions to preserve protein-protein interactions. Based on published methodologies, specific antibodies against HMMR can be used to pull down protein complexes, followed by western blot analysis to detect interacting partners such as FAK, SRC, CD44, and CD47 . Reciprocal co-IP experiments are essential for confirming interactions, where antibodies against the suspected binding partners (e.g., anti-CD44) are used for immunoprecipitation, followed by western blot detection of HMMR . For studying CD44-independent interactions, researchers should consider using CD44 knockout cell lines for co-IP experiments to determine which protein complexes form regardless of CD44 presence . Appropriate negative controls, such as IgG isotype controls and input samples, are critical for validating specific interactions versus non-specific binding.

What controls should be included when using HMMR antibody, HRP conjugated in ELISA assays?

When using HMMR antibody-HRP conjugates in ELISA assays, several critical controls should be incorporated to ensure valid and interpretable results. Negative controls should include wells with no antigen and wells with irrelevant proteins to assess non-specific binding. A positive control with recombinant HMMR protein should be included to confirm antibody functionality . Given that the antibody was raised against a specific region (478-653AA) of human HMMR, researchers should include a peptide competition control using this immunogenic fragment to verify binding specificity . For quantitative assays, a standard curve using purified HMMR protein at known concentrations is essential. Additionally, cross-reactivity controls with related proteins should be performed, especially when working with complex samples. Finally, a dilution series of the HRP-conjugated antibody should be tested to determine the optimal concentration that provides maximum specific signal with minimal background.

How can researchers effectively validate HMMR knockdown or knockout models?

Rigorous validation of HMMR knockdown or knockout models requires a multi-level verification approach. At the genomic level for CRISPR-Cas9 knockout models, PCR genotyping with primers specific to the targeted region (e.g., exon4-exon7 for HMMR) can confirm gene modification . PCR products should be sequenced to verify the exact genetic alteration. At the transcript level, RT-qPCR using validated primers can quantify HMMR mRNA reduction. At the protein level, western blot analysis using specific HMMR antibodies (such as Abcam ab124729, diluted 1:1000) provides direct evidence of protein elimination or reduction . Functional validation through phenotypic assays, such as cell proliferation (colony formation, EdU incorporation) and migration (wound healing) assays, confirms the biological impact of HMMR depletion . For inducible or transient knockdown systems, time-course experiments should be conducted to determine the optimal timepoint for functional studies post-induction.

How can researchers address inconsistent results when studying HMMR in different cancer cell lines?

Inconsistent results across cancer cell lines when studying HMMR may stem from several factors requiring systematic investigation. First, researchers should quantify baseline HMMR expression levels in each cell line using western blot and qRT-PCR, as studies have demonstrated variable HMMR expression across hepatocellular carcinoma cell lines (SNU-449, SMMC7721, HepG2, Huh7, LM3, H22, and Hepa 1-6) . Cell lines with higher HMMR expression (e.g., SMMC7721, Huh7, and LM3) may show more pronounced effects upon knockdown compared to those with lower baseline expression . Second, genetic and epigenetic heterogeneity between cell lines may affect HMMR-associated pathways; therefore, characterizing the status of known HMMR interaction partners (FAK, SRC, CD44, CD47) is essential . Third, differences in culture conditions, passage number, and confluency can impact HMMR function and should be standardized. Finally, when comparing knockdown effects, researchers should ensure equivalent knockdown efficiency across cell lines by optimizing transfection conditions for each line individually.

What approaches can resolve contradictory findings between in vitro and in vivo HMMR studies?

When faced with contradictory findings between in vitro and in vivo HMMR studies, researchers should implement several resolution strategies. First, evaluate whether the in vitro model accurately represents the tumor microenvironment, particularly regarding immune components, as HMMR has been shown to mediate immune evasion through the HMMR-CD47 axis . Consider using co-culture systems with immune cells to better mimic in vivo conditions. Second, assess whether HMMR expression levels in cell lines match those observed in patient tumors by comparing with clinical samples . Third, examine time-dependent effects, as acute HMMR knockdown in vitro may yield different results from sustained knockout in vivo models where compensatory mechanisms may develop. Fourth, use multiple in vivo models, including syngeneic models in HMMR-/- mice and patient-derived xenografts, to capture different aspects of HMMR biology . Finally, perform comprehensive molecular profiling (transcriptomics, proteomics) on both in vitro and in vivo samples to identify context-specific differences in HMMR-regulated pathways.

How should researchers interpret contradictory data regarding HMMR's role in different cancer types?

When interpreting contradictory data on HMMR's role across cancer types, researchers should consider several contextual factors. First, tissue-specific functions of HMMR should be examined, as the protein may interact with different partners in distinct cellular environments. Second, the status of HMMR's binding partners and downstream effectors (FAK, SRC, CD47, CD44) may vary between cancer types, altering pathway outcomes . Third, genetic and epigenetic landscapes differ across cancer types, potentially affecting HMMR regulation and function; therefore, comprehensive genomic analysis should accompany HMMR studies. Fourth, the immune microenvironment varies substantially between cancer types, which may significantly impact HMMR's role in immune evasion . Finally, methodological differences between studies (antibodies used, knockdown approaches, assay conditions) should be carefully evaluated. To resolve contradictions, meta-analyses of HMMR expression and function across multiple datasets and cancer types, coupled with mechanistic studies in diverse cellular contexts, offer the most comprehensive approach.

What factors affect the specificity and sensitivity of HMMR antibody-based detection methods?

Multiple factors influence the specificity and sensitivity of HMMR antibody-based detection methods. The epitope target is crucial; the described HRP-conjugated HMMR antibody targets the 478-653AA region of human HMMR, which may affect detection of specific HMMR isoforms or truncated forms . Cross-reactivity with related proteins, particularly other hyaluronan-binding proteins, should be assessed through appropriate controls. Sample preparation significantly impacts detection; for membrane-associated HMMR, proper cell fractionation and membrane protein extraction protocols are essential, while cytoplasmic HMMR detection requires different sample preparation approaches . Fixation methods for immunohistochemistry or immunofluorescence can affect HMMR epitope accessibility; paraformaldehyde typically preserves HMMR structure while maintaining antigenicity. Signal amplification systems must be optimized for HRP-conjugated antibodies, with substrate selection affecting sensitivity, linear range, and signal-to-noise ratio. Finally, biological variations in HMMR glycosylation or post-translational modifications across different cell types may affect antibody recognition and should be considered when comparing results across experimental systems.

How might HMMR antibodies be utilized to develop novel therapeutic approaches for cancer?

HMMR antibodies hold significant potential for novel cancer therapeutic development through multiple mechanisms. They could be developed as direct targeting agents that disrupt HMMR's interaction with CD47, thereby blocking the "don't eat me" signal and enhancing macrophage-mediated phagocytosis of cancer cells . HMMR-targeting antibodies might also interrupt the HMMR-FAK/SRC complex formation, inhibiting downstream NF-κB signaling that sustains CD47 expression . Additionally, antibody-drug conjugates utilizing HMMR antibodies could deliver cytotoxic payloads specifically to HMMR-overexpressing tumor cells while sparing normal tissues. Perhaps most promisingly, HMMR antibodies might function as immune checkpoint inhibitor adjuvants, as research has demonstrated that targeting HMMR can enhance anti-PD-1 treatment efficiency by recruiting CD8+ T cells to the tumor microenvironment . Pre-clinical models combining HMMR knockout with anti-PD-1 therapy have already shown enhanced treatment efficacy, suggesting HMMR antibody-based approaches could similarly improve immunotherapy outcomes.