RMND5A Antibody

What is RMND5A and what is its primary function in cellular processes?

RMND5A (Required For Meiotic Nuclear Division 5 Homolog A) functions as a core component of the CTLH E3 ubiquitin-protein ligase complex. This complex plays a critical role in the ubiquitin-proteasome system, which is essential for protein quality control in cells. RMND5A specifically assists in the selective acceptance of ubiquitin from UBE2H and mediates the ubiquitination of target proteins, marking them for subsequent proteasomal degradation .

The E3 ubiquitin ligase activity of RMND5A is particularly important for the degradation of specific transcription factors such as HBP1. Research has demonstrated that both MAEA and RMND5A are required for the catalytic activity of the CTLH E3 ubiquitin-protein ligase complex, and this activity is necessary for normal cell proliferation . Importantly, RMND5A is not involved in all protein degradation pathways; for example, it is not required for the degradation of enzymes involved in gluconeogenesis, such as FBP1 .

What is the molecular weight of RMND5A and how does this information help in Western blot analysis?

When designing Western blot experiments, researchers should anticipate this molecular weight range and be aware that post-translational modifications or protein processing might affect the apparent molecular weight. Some researchers have reported difficulties in detecting RMND5A cleanly in certain cell lines, with observations of non-specific bands , which necessitates careful optimization of experimental conditions and proper inclusion of controls.

What criteria should be considered when selecting an RMND5A antibody for specific research applications?

When selecting an RMND5A antibody, researchers should consider several key factors to ensure optimal experimental outcomes:

• Application compatibility: Different antibodies are validated for specific applications such as Western blot (WB), immunohistochemistry (IHC), or ELISA. For example, antibody ABIN7246598 is recommended for IHC at dilutions of 1:40-1:200 and ELISA at 1:5000-1:10000 , while antibody 17559-1-AP is validated for WB at 1:500-1:2000 and IHC at 1:50-1:500 .

• Species reactivity: Ensure the antibody recognizes RMND5A in your species of interest. Available antibodies have different reactivity profiles - some recognize human RMND5A exclusively, while others cross-react with mouse and rat RMND5A .

• Clonality: Consider whether a polyclonal or monoclonal antibody is more suitable for your application. Most available RMND5A antibodies are rabbit polyclonal antibodies .

• Epitope region: Some antibodies target specific regions of RMND5A, such as the C-terminus (AA 300-327) or other specific amino acid sequences (e.g., AA 1-391, AA 200-270) . This is crucial if you're studying specific domains or if certain regions might be masked in your experimental system.

• Validation data: Review existing validation data, including Western blot images showing band specificity and IHC staining patterns. Published studies utilizing the antibody provide valuable information about performance in real research contexts .

How can I validate the specificity of an RMND5A antibody in my experimental system?

Validating antibody specificity is crucial for ensuring reliable results. For RMND5A antibodies, a comprehensive validation approach should include:

• Positive and negative controls: Use cell lines known to express RMND5A (such as Jurkat or K-562 cells) as positive controls . For negative controls, consider using CRISPR knockout cell lines, although researchers have reported challenges with RMND5A knockouts in HEK293T cells .

• Multiple detection methods: Validate expression using complementary techniques (e.g., WB, IHC, and RT-qPCR) to confirm consistent patterns of expression across methods.

• Knockdown/overexpression experiments: Compare antibody detection in systems with normal, reduced (siRNA/shRNA), and overexpressed RMND5A levels. Research has utilized RMND5A overexpression constructs in pancreatic cancer cell lines (AsPC-1 and PANC-1) .

• Molecular weight verification: Confirm that the detected band appears at the expected molecular weight of 40-44 kDa .

• Peptide competition assay: Pre-incubate the antibody with the immunogen peptide to demonstrate specific binding, which should abolish or significantly reduce the signal.

What are the optimal protocols for using RMND5A antibodies in immunohistochemistry?

For successful immunohistochemistry (IHC) with RMND5A antibodies, researchers should follow these methodological guidelines:

How should I optimize Western blot conditions for detecting RMND5A protein?

Optimizing Western blot conditions for RMND5A detection requires attention to several technical aspects:

• Sample preparation: Use appropriate lysis buffers that effectively solubilize membrane-associated proteins while preserving epitope integrity. Include protease inhibitors to prevent degradation.

• Protein loading: Load 20-40 μg of total protein per lane, with Jurkat or K-562 cell lysates serving as positive controls .

• Gel percentage: Use 10-12% SDS-PAGE gels for optimal resolution of RMND5A's 40-44 kDa band.

• Transfer conditions: Optimize transfer time and voltage for proteins in this molecular weight range (typically 100V for 60-90 minutes).

• Blocking: Use 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature to minimize background.

• Antibody dilution: For antibody 17559-1-AP, use a dilution of 1:500-1:2000 . Incubate overnight at 4°C for optimal results.

• Washing: Perform thorough washing steps (5-6 times, 5 minutes each) with TBST to reduce background.

• Detection system: Use an appropriate secondary antibody (anti-rabbit IgG) and detection method (ECL, fluorescence) compatible with your imaging system.

• Exposure time: Start with short exposures and gradually increase to find the optimal signal-to-noise ratio, as RMND5A expression levels may vary between cell types.

Why might I be seeing multiple non-specific bands when using RMND5A antibodies in Western blots?

Multiple non-specific bands in RMND5A Western blots can occur for several reasons, with researchers reporting specific challenges with RMND5A detection :

• Antibody specificity issues: Some RMND5A antibodies have been reported to show numerous non-specific bands, particularly in HEK293T cells . This may be inherent to the antibody or related to the complex nature of the ubiquitin-proteasome system in which RMND5A functions.

• Cross-reactivity with related proteins: RMND5A shares sequence homology with other proteins involved in ubiquitination pathways, which may lead to cross-reactivity.

• Post-translational modifications: RMND5A may undergo ubiquitination, phosphorylation, or other modifications that change its molecular weight and create multiple bands.

• Protein degradation: Improper sample handling or insufficient protease inhibition can lead to degradation products appearing as multiple bands.

To address these issues:

• Optimize blocking conditions (try both milk and BSA at different concentrations)

• Test different antibody concentrations and incubation times

• Use freshly prepared samples with complete protease inhibitor cocktails

• Consider using genetic approaches (siRNA knockdown or CRISPR knockout) to confirm band specificity

• Compare results across different cell lines, as some may express RMND5A more clearly than others

• Try alternative RMND5A antibodies that target different epitopes

What controls should be included when studying RMND5A expression in cancer research?

When investigating RMND5A in cancer research, comprehensive controls are essential:

• Tissue/cell type controls:

  • Positive controls: Include Jurkat cells and K-562 cells, which are validated to express RMND5A

  • Normal tissue controls: Always compare cancer samples with matched normal tissues to establish baseline expression

  • Multiple cancer types: If possible, include various cancer types as RMND5A expression varies (highly expressed in pancreatic adenocarcinoma, stomach adenocarcinoma, and thymoma)

• Expression modulation controls:

  • Overexpression system: The pHS-AVC vector system has been successfully used for RMND5A overexpression

  • Knockdown systems: siRNA or shRNA targeting RMND5A

  • miRNA modulation: miR-590-5p mimics can be used to downregulate RMND5A expression

• Functional controls:

  • Migration assays: Track changes in cell migration, as RMND5A overexpression has been shown to significantly increase migration in pancreatic cancer cell lines

  • Survival analysis: Correlate RMND5A expression with patient survival data to validate clinical relevance

• Technical controls:

  • Loading controls: Standard proteins (β-actin, GAPDH) to normalize expression levels

  • Antibody specificity: Secondary-only controls to assess background

  • Multiple antibodies: If possible, use antibodies targeting different epitopes of RMND5A

How does RMND5A expression correlate with cancer progression and patient outcomes?

RMND5A expression has significant implications for cancer progression and patient outcomes, particularly in pancreatic adenocarcinoma (PAAD):

What experimental approaches can be used to study the interaction between miR-590-5p and RMND5A?

To investigate the miR-590-5p/RMND5A regulatory axis, researchers can employ these methodological approaches:

• Expression correlation analysis:

  • RT-qPCR to measure both miR-590-5p and RMND5A expression levels in the same samples

  • Western blot analysis to confirm protein-level changes

  • In silico analysis using cancer databases to examine expression correlations across larger patient cohorts

• Direct interaction validation:

  • Dual-luciferase reporter assay using constructs containing the RMND5A 3'UTR (wild-type and miR-590-5p binding site mutants)

  • RNA immunoprecipitation (RIP) assays to confirm the presence of RMND5A mRNA in miRNA-induced silencing complexes

• Functional studies:

  • Transfection of miR-590-5p mimics (100 nM concentration has been validated) to downregulate RMND5A

  • Co-transfection experiments with miR-590-5p mimics and RMND5A overexpression constructs to demonstrate rescue effects

  • Wound-healing assays to assess migration potential, as this has been shown to be regulated by the miR-590-5p/RMND5A axis

• Mechanistic investigations:

  • Analysis of the 3'UTR conservation among mammals, as both miR-590-5p and RMND5A 3'UTR are highly conserved

  • ClipSeq analysis to validate the physical interaction between miR-590-5p and RMND5A mRNA

  • Target site validation through site-directed mutagenesis of the miR-590-5p binding site in the RMND5A 3'UTR

• Translational approaches:

  • In vivo studies using xenograft models with modulated miR-590-5p/RMND5A expression

  • Correlation of miR-590-5p/RMND5A expression ratios with patient outcomes in clinical samples

How can RMND5A antibodies be used to investigate the role of RMND5A in the ubiquitin-proteasome system?

Investigating RMND5A's role in the ubiquitin-proteasome system requires specialized experimental approaches:

• Co-immunoprecipitation (Co-IP) studies:

  • Use RMND5A antibodies to precipitate native protein complexes

  • Identify interaction partners within the CTLH E3 ubiquitin-protein ligase complex

  • Western blot analysis for known components (MAEA, UBE2H) to confirm complex formation

  • Mass spectrometry to identify novel interaction partners or substrates

• Ubiquitination assays:

  • In vitro ubiquitination assays using purified components

  • In vivo ubiquitination assays with co-expression of RMND5A and potential substrates

  • Detection of ubiquitinated proteins using anti-ubiquitin antibodies following RMND5A immunoprecipitation

  • Analysis of substrate (e.g., HBP1) ubiquitination in the presence/absence of RMND5A

• Proteasome inhibition studies:

  • Treatment of cells with proteasome inhibitors (MG132, bortezomib)

  • Comparison of substrate levels with/without RMND5A expression

  • Pulse-chase experiments to determine protein half-life changes dependent on RMND5A

• Structural studies:

  • Immunofluorescence or immunohistochemistry to determine subcellular localization

  • Co-localization studies with other ubiquitin-proteasome system components

  • FRET or PLA (Proximity Ligation Assay) to demonstrate direct physical interactions in situ

• Functional rescue experiments:

  • RMND5A knockdown followed by expression of wild-type or mutant RMND5A

  • Analysis of cellular proliferation, which requires catalytic activity of the CTLH complex

  • Assessment of specific substrate degradation in rescue systems