ERH Antibody Pair

What is ERH and why is it significant in research?

ERH (Enhancer of rudimentary homolog) is a protein that plays a crucial role in cell cycle regulation and has been implicated in various cancer types. Recent research has demonstrated that ERH is upregulated in lung adenocarcinoma (LUAD) and is associated with worse survival prognosis in cancer patients. The significance of ERH lies in its potential involvement in promoting epithelial-mesenchymal transition (EMT) and cell migration, which are critical processes in cancer progression. Furthermore, ERH has been linked to an immunosuppressive tumor microenvironment, making it an important target for cancer research and potential therapeutic development .

What types of ERH antibodies are currently available for research applications?

Several types of ERH antibodies are available for research applications, including recombinant monoclonal antibodies. For instance, the Rabbit Recombinant Monoclonal ERH antibody [EPR10830(B)] is suitable for multiple applications including immunohistochemistry with paraffin-embedded tissues (IHC-P), immunoprecipitation (IP), Western blotting (WB), and immunocytochemistry/immunofluorescence (ICC/IF). This particular antibody has been validated for reactivity with human, mouse, and rat samples, providing versatility for comparative studies across species . When selecting an ERH antibody, researchers should consider the specific application requirements, species reactivity, and validation status of the antibody for their experimental system.

How can I validate the specificity of an ERH antibody?

Validating antibody specificity is crucial for obtaining reliable results. For ERH antibodies, validation can be performed through several approaches:

• Western blot validation: Confirm the detection of a single band at the expected molecular weight (approximately 12 kDa for ERH) .

• Positive and negative control tissues/cells: Use samples known to express ERH (such as A549 or T-47D cells) and compare with samples where ERH has been knocked down using siRNA .

• Immunohistochemistry controls: Compare staining patterns between tumor and adjacent non-tumor tissues, as ERH has been shown to be upregulated in certain cancers like LUAD .

• Blocking peptide competition: Pre-incubate the antibody with purified ERH protein to demonstrate specific binding.

• Orthogonal validation: Correlate protein detection with mRNA expression data from techniques like RT-PCR or RNA-seq.

A comprehensive validation should include at least two independent methods to confirm antibody specificity and reproducibility across different experimental conditions.

What are the optimal protocols for ERH antibody use in Western blotting?

For optimal Western blot results with ERH antibodies, follow these methodological guidelines:

• Sample preparation: Prepare cell lysates from relevant cell lines (e.g., T-47D, SH-SY5Y, A549, A431) using a complete lysis buffer containing protease inhibitors.

• Protein loading: Load approximately 10-20 μg of total protein per lane.

• Separation: Use 15-20% SDS-PAGE gels to effectively resolve the low molecular weight ERH protein (approximately 12 kDa).

• Transfer: Employ a semi-dry or wet transfer system with methanol-containing transfer buffer for efficient transfer of small proteins.

• Blocking: Block membranes with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute ERH antibody to 1/1000 in blocking buffer and incubate overnight at 4°C .

• Secondary antibody: Use an appropriate HRP-conjugated secondary antibody at 1/5000-1/10000 dilution.

• Detection: Develop using enhanced chemiluminescence (ECL) with appropriate exposure times.

The expected result should be a single band at approximately 12 kDa, corresponding to the predicted molecular weight of ERH .

How can I effectively use ERH antibodies for immunohistochemistry?

For optimal immunohistochemical detection of ERH, the following protocol is recommended:

• Tissue preparation: Use formalin-fixed, paraffin-embedded tissue sections (4-6 μm thickness).

• Antigen retrieval: Perform heat-mediated antigen retrieval with citrate buffer (pH 6.0) before commencing with the IHC staining protocol .

• Blocking: Block endogenous peroxidase activity with hydrogen peroxide and non-specific binding with appropriate serum.

• Primary antibody: Dilute ERH antibody to approximately 1/250 in blocking solution and incubate overnight at 4°C .

• Detection system: Use a polymer-based detection system for enhanced sensitivity.

• Counterstaining: Counterstain with hematoxylin to visualize tissue architecture.

• Controls: Include positive controls (e.g., human cervical carcinoma or prostate tissue) and negative controls (primary antibody omitted) .

Researchers should expect to observe differential staining between tumor and non-tumor tissues, with enhanced ERH expression typically observed in tumor tissues, particularly in lung adenocarcinoma samples .

What are the best approaches for using ERH antibodies in immunoprecipitation experiments?

For effective immunoprecipitation of ERH protein:

• Cell lysis: Prepare cell lysates from appropriate cell lines (e.g., A549 cells) using a mild lysis buffer containing protease inhibitors.

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Antibody binding: Incubate cleared lysates with ERH antibody at a dilution of approximately 1/10 .

• Immunoprecipitation: Add protein A/G beads and incubate with gentle rotation overnight at 4°C.

• Washing: Perform multiple washes with lysis buffer to remove non-specifically bound proteins.

• Elution and analysis: Elute bound proteins by boiling in SDS sample buffer and analyze by Western blotting.

This approach can be particularly useful for studying ERH interactions with other proteins, such as SNRPG, which has been identified as a potential interaction partner .

How can ERH antibodies be used to investigate its role in cancer progression?

ERH antibodies can be instrumental in investigating the role of ERH in cancer progression through several advanced applications:

• Expression profiling: Compare ERH expression levels between tumor and adjacent non-tumor tissues using IHC to correlate with clinical outcomes. Studies have shown that high ERH expression is associated with worse survival prognosis in lung adenocarcinoma .

• Functional assays after manipulation: Combine ERH antibody-based detection with siRNA knockdown experiments to assess how ERH affects:

  • Cell migration (Transwell and wound healing assays)

  • EMT marker expression (E-cadherin, N-cadherin, vimentin)

  • Cell proliferation

• Protein-protein interaction studies: Use co-immunoprecipitation with ERH antibodies to identify and validate interaction partners such as SNRPG and other components of the spliceosome machinery .

• Mechanism investigation: Employ chromatin immunoprecipitation (ChIP) with ERH antibodies to identify potential DNA binding sites and regulatory mechanisms.

These findings suggest that ERH predominantly affects cancer cell migration and EMT rather than proliferation .

What considerations should be made when studying ERH in different cancer types?

When studying ERH across different cancer types, researchers should consider:

• Expression variability: ERH expression may vary significantly between cancer types. In lung adenocarcinoma, ERH is upregulated compared to adjacent non-tumor tissue , but this pattern should be independently verified for each cancer type.

• Prognostic significance: The association between ERH expression and patient survival may differ by cancer type. In LUAD, high ERH expression correlates with worse survival outcomes .

• Cell line selection: Choose appropriate cell lines that represent the cancer type of interest. For lung cancer studies, A549 and CL1-0 cell lines have been successfully used .

• Pathway interactions: ERH may interact with different pathways in different cancer contexts. In LUAD, ERH positively correlates with cell cycle, DNA metabolic processes, and S phase .

• Immune microenvironment impact: ERH expression has been associated with an immunosuppressive tumor microenvironment in LUAD, with increased infiltration of myeloid-derived suppressor cells (MDSCs) and natural regulatory T cells (nTregs) . This association should be evaluated in each cancer type.

• Antibody validation: Re-validate ERH antibodies for specific cancer types, as protein modifications or interactions might affect epitope accessibility.

How can I use ERH antibodies to investigate its interaction with SNRPG?

To investigate the ERH-SNRPG interaction, researchers can employ the following approaches using ERH antibodies:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate ERH using anti-ERH antibodies and probe for SNRPG in the precipitate.

  • Perform the reverse experiment by immunoprecipitating SNRPG and probing for ERH.

  • Use appropriate controls including IgG controls and cell lysates with ERH knockdown.

• Proximity ligation assay (PLA):

  • Use ERH and SNRPG antibodies from different species.

  • Employ PLA to visualize and quantify the interaction in situ within cells.

• Co-localization studies:

  • Perform immunofluorescence with ERH and SNRPG antibodies.

  • Analyze co-localization using confocal microscopy and quantitative image analysis.

• Expression correlation:

  • Use ERH and SNRPG antibodies for IHC on tissue microarrays.

  • Correlate expression patterns across multiple patient samples.

  • Current research indicates that in 7 out of 10 patients with LUAD, higher expression of ERH was accompanied by elevated SNRPG expression .

• Functional studies:

  • Employ RNA interference to knockdown ERH or SNRPG.

  • Use respective antibodies to monitor expression changes in the partner protein.

  • Assess functional outcomes in terms of RNA splicing, cell migration, and EMT.

The ERH-SNRPG interaction appears to be particularly relevant for LUAD progression, as the survival impact of ERH was only observed in cells with high SNRPG expression, suggesting an SNRPG-dependent modulating process .

How do I address inconsistent results with ERH antibodies?

When encountering inconsistent results with ERH antibodies, consider these systematic troubleshooting approaches:

• Antibody validation issues:

  • Re-validate antibody specificity using positive and negative controls.

  • Consider testing a different lot or alternative ERH antibody.

  • Verify the antibody concentration and storage conditions.

• Protocol optimization:

  • For Western blotting: Adjust protein loading, transfer conditions, antibody dilution, and incubation times.

  • For IHC: Optimize antigen retrieval methods, consider different fixation protocols, and test various antibody dilutions.

  • For immunoprecipitation: Modify lysis buffer composition, adjust antibody-to-lysate ratios, and vary wash stringency.

• Sample-related issues:

  • Ensure proper sample handling and storage to prevent protein degradation.

  • For cell lines, verify ERH expression levels using RT-qPCR before protein analysis.

  • For tissue samples, assess tissue quality and fixation consistency.

• Technical considerations:

  • For Western blotting of ERH (12 kDa), ensure your gel percentage and transfer conditions are optimized for small proteins.

  • For IHC, perform heat-mediated antigen retrieval with citrate buffer pH 6 before staining .

  • For functional studies, confirm knockdown efficiency before interpreting phenotypic results.

• Biological variability:

  • Consider the cell cycle stage, as ERH may have cell cycle-dependent expression patterns.

  • Account for potential post-translational modifications that might affect antibody recognition.

  • In cancer studies, tumor heterogeneity may contribute to variable ERH expression within the same sample.

How can I analyze ERH expression data in relation to clinical outcomes?

To effectively analyze ERH expression data in relation to clinical outcomes:

What are emerging applications for ERH antibodies in cancer research?

Emerging applications for ERH antibodies in cancer research include:

• ERH as a therapeutic target: Developing and testing potential drugs targeting ERH would benefit from high-quality antibodies for target validation and mechanism studies. Current evidence suggests that ERH could be a promising target for LUAD treatment .

• Liquid biopsy development: ERH antibodies could potentially be used to detect circulating tumor cells or exosomes expressing ERH, providing a non-invasive method to monitor disease progression.

• Precision medicine applications: ERH expression analysis might help stratify patients for targeted therapies or immunotherapies, particularly given the association between ERH expression and an immunosuppressive tumor microenvironment .

• Combination with spatial transcriptomics: Integrating ERH antibody-based protein detection with spatial transcriptomics could provide insights into the tumor microenvironment and heterogeneity.

• ERH-SNRPG interaction targeting: The development of therapeutic strategies targeting the ERH-SNRPG interaction could be facilitated by antibodies that specifically recognize this interaction interface .

• Immune response modulation: Given ERH's association with immunosuppressive cell infiltration, antibodies could help investigate how ERH modulates the immune response in the tumor microenvironment .

• RNA splicing regulation: ERH appears to be involved in RNA splicing processes, potentially through interaction with SNRPG and other spliceosome components. Antibodies would be valuable tools for dissecting these mechanisms .

These emerging applications highlight the potential of ERH antibodies beyond basic research, positioning them as valuable tools for translational cancer research and potential therapeutic development.