HMGN4 Antibody

What is HMGN4 and why is it significant in research?

HMGN4 (high mobility group nucleosomal binding domain 4) is a 90 amino acid DNA-binding protein that belongs to the HMGN family and localizes in the nucleus. It is the latest discovered member of the HMGN protein family and is uniquely encoded by a retrogene . Unlike most intronless retrogenes which do not encode proteins, HMGN4 is expressed in human cells and has been verified through western analysis using HMGN4-specific antibodies . The protein has gained significant research interest due to its elevated expression in several cancers, particularly thyroid cancers, suggesting its potential role in oncogenesis .

What are the standard applications for HMGN4 antibodies?

HMGN4 antibodies are primarily used in the following standard laboratory applications:

These applications allow researchers to detect HMGN4 protein expression, cellular localization, and interactions in experimental settings . It is recommended that researchers titrate the antibody in each testing system to obtain optimal results, as performance can be sample-dependent .

What is the molecular weight of HMGN4 and why does it differ from calculated predictions?

While the calculated molecular weight of HMGN4 based on its amino acid sequence is approximately 10 kDa, the observed molecular weight in experimental conditions is typically around 15 kDa . This discrepancy is attributed to post-translational modifications of the protein. When conducting western blot analysis, researchers should expect to observe bands at approximately 15 kDa rather than at the theoretical 10 kDa position . Understanding this difference is crucial for accurate interpretation of experimental results.

How should I optimize HMGN4 antibody usage for Western blotting?

For optimal Western blot results with HMGN4 antibodies, consider the following methodological approach:

• Sample preparation: For effective HMGN4 detection, prepare perchloric acid (PCA) extracts of your samples, as this method efficiently extracts nuclear proteins including HMG family proteins .

• Dilution optimization: Begin with a middle-range dilution (e.g., 1:1000) and adjust based on signal intensity. The recommended range is typically 1:500-1:2000 .

• Blocking conditions: Use 5% nonfat milk in 1× phosphate-buffered saline to minimize background .

• Detection method: For enhanced sensitivity, use chemiluminescence detection following the manufacturer's recommendations (e.g., SuperSignal West kits) .

• Size verification: Confirm that your detected band appears at approximately 15 kDa, which is the observed molecular weight of HMGN4, rather than the calculated 10 kDa .

• Controls: Include both positive controls (known HMGN4-expressing cells such as HeLa) and negative controls to validate antibody specificity .

What are the recommended protocols for immunofluorescence experiments using HMGN4 antibodies?

When conducting immunofluorescence experiments with HMGN4 antibodies:

• Cell preparation: Culture cells on appropriate coverslips or slides and fix using a method that preserves nuclear architecture (e.g., 4% paraformaldehyde).

• Permeabilization: Since HMGN4 is a nuclear protein, effective nuclear permeabilization is crucial for antibody access.

• Antibody dilution: Use the recommended dilution range of 1:400-1:1600 for primary HMGN4 antibody incubation .

• Nuclear counterstain: Include DAPI or a similar nuclear stain to confirm nuclear localization of HMGN4.

• Controls: Include cells with known HMGN4 expression (such as HeLa cells) as positive controls .

• Visualization: Expected pattern should show primarily nuclear localization consistent with HMGN4's function as a DNA-binding protein .

Following standardized protocols available from antibody manufacturers can help ensure reproducible results .

How can HMGN4 antibodies be used to investigate its role in cancer research?

HMGN4 antibodies serve as valuable tools in cancer research, particularly in studying thyroid carcinogenesis:

• Expression analysis: Use HMGN4 antibodies in immunohistochemistry or western blotting to compare expression levels between normal and cancerous tissues. Research indicates that HMGN4 expression is elevated in several cancers, including thyroid cancers .

• Functional studies: Employ HMGN4 antibodies in chromatin immunoprecipitation (ChIP) assays to identify genomic regions bound by HMGN4, providing insights into its regulatory roles.

• Prognostic marker investigation: Study correlations between HMGN4 expression levels (detected via antibodies) and patient outcomes to assess its potential as a diagnostic or prognostic marker .

• Therapeutic target assessment: Use HMGN4 antibodies to monitor protein levels following experimental therapeutic interventions targeted at reducing HMGN4 expression or activity .

• Mechanistic investigations: Research shows that HMGN4 overexpression affects genes such as Atm, Atrx, and Brca2, and elevates levels of DNA damage marker γH2AX . HMGN4 antibodies can be used to study these interactions and pathways.

What are the considerations for using HMGN4 antibodies in combination with other HMGN family protein detection?

When designing experiments to detect multiple HMGN family proteins:

• Antibody specificity: Ensure your HMGN4 antibody has been validated for specificity against other HMGN family members. For example, some HMGN4 antibodies are raised against peptides spanning amino acids 67-82 of human HMGN4, which provides specificity .

• Cross-reactivity assessment: Test for potential cross-reactivity with other HMGN proteins, particularly HMGN1 and HMGN2, which share structural similarities.

• Multiplexing strategies: For co-detection, select antibodies raised in different host species or use directly conjugated antibodies with distinct fluorophores.

• Sequential immunoprecipitation: Consider sequential IP approaches to study interactions between different HMGN family members.

• Comparative analysis: Use western blotting with specific antibodies against different HMGN family members to compare expression patterns across various cell types or conditions .

How can I verify the specificity of my HMGN4 antibody?

To ensure antibody specificity and minimize false-positive results:

• Immunogen verification: Confirm that the antibody was raised against a specific HMGN4 sequence. For example, antibodies elicited by peptides spanning amino acids 67-82 of human HMGN4 have demonstrated specificity .

• Molecular weight validation: Verify that detected bands appear at the expected 15 kDa rather than the calculated 10 kDa, which is a distinguishing characteristic of HMGN4 .

• Knockdown/knockout validation: Test the antibody in HMGN4 knockdown or knockout samples, where signal should be significantly reduced or absent.

• Competitive peptide blocking: Pre-incubate the antibody with the immunizing peptide prior to use in experiments; this should abolish specific binding.

• Cross-reactivity testing: Test against recombinant HMGN family proteins to ensure the antibody does not detect related family members.

• Multiple antibody validation: When possible, confirm findings using multiple antibodies targeting different epitopes of HMGN4.

What are common issues when using HMGN4 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with HMGN4 antibodies:

• High background in Western blots:

  • Solution: Optimize blocking conditions using 5% nonfat milk in PBS , increase washing steps, and ensure antibody dilution is appropriate (1:500-1:2000) .

• Weak or no signal:

  • Solution: For nuclear proteins like HMGN4, ensure proper extraction methods (such as perchloric acid extraction) and consider signal enhancement systems.

• Multiple bands or unexpected band sizes:

  • Solution: Verify sample preparation methods, use fresh samples to prevent degradation, and confirm that observed bands match the expected 15 kDa size for HMGN4 .

• Poor reproducibility between experiments:

  • Solution: Standardize protocols, maintain consistent antibody storage conditions (store at -20°C, avoid repeated freeze/thaw cycles) , and follow manufacturer's recommendations for aliquoting.

• Inconsistent immunofluorescence staining:

  • Solution: Ensure adequate permeabilization for nuclear access, optimize antibody dilution (1:400-1:1600) , and use appropriate fixation methods.

How is HMGN4 antibody being used in immunotherapy research?

While HMGN4-specific immunotherapy applications are still emerging, research on related family members provides insights into potential directions:

• Combined immunotherapy approaches: Research with HMGN1 (a related family member) shows promising results when used in combination with anti-CD4 depleting antibody for cancer treatment . Similar approaches might be explored with HMGN4, particularly in thyroid cancers where its expression is elevated.

• Immunomodulatory mechanisms: HMGN1 treatment promotes expansion of CD8+ T cell populations and reduces co-inhibitory molecules to counteract T cell exhaustion . HMGN4 antibodies can help investigate whether similar mechanisms apply to HMGN4.

• Therapeutic target potential: Given the role of HMGN4 in tumorigenicity, antibodies can help validate it as a therapeutic target, particularly in thyroid cancers .

• Biomarker development: HMGN4 antibodies may facilitate the development of diagnostic tests to identify cancers with elevated HMGN4 expression, potentially guiding treatment decisions .

• Mechanism elucidation: HMGN4 antibodies can help elucidate the relationship between HMGN4 expression and changes in genes like Atm, Atrx, and Brca2, which are affected by HMGN4 overexpression .

What are the latest findings regarding HMGN4 structure-function relationships that impact antibody selection?

Recent structural insights have implications for antibody selection and experimental design:

• Domain-specific antibodies: HMGN4 contains a nucleosome-binding domain that mediates its interaction with chromatin. Antibodies targeting different domains may provide insights into specific functions.

• Post-translational modifications: The discrepancy between calculated (10 kDa) and observed (15 kDa) molecular weights suggests significant post-translational modifications . Antibodies that specifically recognize modified forms may be valuable for studying HMGN4 regulation.

• Species conservation considerations: When selecting antibodies for cross-species studies, consider that HMGN4 is a retrogene with potentially different conservation patterns than other HMGN family members .

• Functional epitopes: Antibodies targeting functional epitopes might interfere with HMGN4's DNA-binding capacity or protein-protein interactions, offering tools for functional blockade experiments.

• Nuclear localization: HMGN4's nuclear localization means that antibodies used for live-cell applications must be able to access nuclear compartments, influencing experimental design.