DYNLRB2 Antibody

What is DYNLRB2 and what is its biological function?

DYNLRB2 functions as a non-catalytic accessory component of the cytoplasmic dynein 1 complex. It plays crucial roles in linking dynein to cargos and adapter proteins that regulate dynein function. DYNLRB2 is particularly upregulated during male meiosis and is indispensable for spindle formation in meiosis I. Studies have shown that DYNLRB2 inhibits pericentriolar material (PCM) fragmentation through two distinct pathways: suppressing premature centriole disengagement and targeting Nuclear Mitotic Apparatus protein (NuMA) to spindle poles . Cytoplasmic dynein 1 acts as a motor for the intracellular retrograde motility of vesicles and organelles along microtubules . DYNLRB2 expression has also been found to be significantly down-regulated in hepatocellular carcinoma patients, suggesting a potential role as a biomarker .

What applications are DYNLRB2 antibodies validated for?

DYNLRB2 antibodies have been validated for multiple research applications with specific optimization parameters:

These recommendations provide starting points for assay optimization, though actual working concentrations should be determined by the researcher through careful titration . Most commercially available DYNLRB2 antibodies have been validated to show reactivity with human, mouse, and rat samples .

What is the optimal sample preparation method for detecting DYNLRB2 in Western blots?

For optimal detection of DYNLRB2 in Western blot applications, researchers should consider the protein's molecular characteristics. DYNLRB2 has a calculated molecular weight of 6 kDa but shows an observed molecular weight of approximately 11 kDa on SDS-PAGE gels . This discrepancy suggests possible post-translational modifications or structural features affecting migration.

For effective detection:

• Use high-percentage (12-15%) SDS-PAGE gels to properly resolve this low molecular weight protein

• Include protease inhibitors in lysis buffers to prevent degradation

• Transfer to PVDF membranes (rather than nitrocellulose) for better retention of small proteins

• Use mouse or rat testis tissue as positive controls for validation

• Apply longer blocking times (at least 1 hour) to reduce background

• Consider wet transfer methods at lower voltages for extended periods for optimal protein transfer

How should researchers troubleshoot non-specific binding when using DYNLRB2 antibodies?

When encountering non-specific binding with DYNLRB2 antibodies, researchers should implement a systematic troubleshooting approach:

• Validate antibody specificity using Dynlrb2 knockdown models, as described in MLV infection studies

• Increase washing stringency by using PBS-T with higher detergent concentrations (0.1-0.3% Tween-20)

• Optimize blocking conditions using different blocking agents (BSA, non-fat milk, commercial blockers)

• Consider using peptide competition assays with the immunizing peptide

• Test different fixation protocols for immunostaining applications

• Perform antibody titration to determine optimal concentration

• Use alternative detection systems if high background persists

These approaches have proven effective in studies examining DYNLRB2's role in viral trafficking, where specific detection was crucial for tracking its interactions with viral components .

How can researchers distinguish between DYNLRB1 and DYNLRB2 in experimental systems?

Distinguishing between the closely related paralogs DYNLRB1 and DYNLRB2 requires specific methodological considerations:

• Expression pattern analysis: DYNLRB2 is primarily testis-upregulated, while DYNLRB1 is ubiquitously expressed in mitotic cells . Examining tissue-specific expression can help differentiate the two proteins.

• Functional analysis: DYNLRB2 plays specialized roles in male meiosis and spindle formation in meiosis I, while DYNLRB1 maintains spindle bipolarity in mitotic cells by targeting NuMA and suppressing centriole overduplication .

• Immunostaining approaches:

  • Use highly specific antibodies targeting non-conserved epitopes

  • Perform sequential immunostaining with paralog-specific antibodies

  • Include appropriate positive controls (testis tissue for DYNLRB2)

• Knockout/knockdown validation: Generate and utilize DYNLRB1 and DYNLRB2 specific knockout/knockdown models to confirm antibody specificity and functional differences .

Research has shown that these two paralogs form distinct dynein complexes used separately in mitotic and meiotic spindle formations, with both having NuMA as a common target .

What methodological approaches can be used to study DYNLRB2's role in viral infection mechanisms?

Studies have demonstrated that DYNLRB2 is essential for murine leukemia virus (MLV) traffic along microtubules and nuclear localization . Researchers investigating DYNLRB2's role in viral infection can employ these methodological approaches:

• Generation of stable Dynlrb2 knockdown cell lines:

  • Using siRNA or shRNA approaches to significantly decrease Dynlrb2 mRNA levels

  • Confirm knockdown efficiency using RT-qPCR

  • Include rescue experiments with HA-tagged Dynlrb2 expression

• Visualization of viral trafficking:

  • Use fluorescently tagged viral particles (e.g., GFP-p12 fusion proteins)

  • Track preintegration complex (PIC) movement to the nucleus at multiple time points (4, 8, and 12 hours post-infection)

  • Perform immunofluorescence with anti-GFP antibodies to enhance detection sensitivity

• Quantitative analysis:

  • Measure the net speed of fluorescently labeled incoming particles in live cells

  • Quantify the percentage of viral particles reaching the nucleus versus remaining in the cytoplasm

  • Compare trafficking patterns between control, knockdown, and rescue conditions

Research has shown that silencing Dynlrb2 significantly reduces MLV infection by impairing cytoplasmic traffic and nuclear entry of the viral preintegration complex .

How can DYNLRB2 antibodies be optimized for studying its role in meiotic spindle formation?

To investigate DYNLRB2's critical role in meiotic spindle formation, researchers should implement these specialized immunofluorescence approaches:

• Sample preparation optimization:

  • Use fresh or properly preserved testis tissue sections

  • Test different fixatives to maintain both protein localization and spindle structure

  • Consider specialized fixation for meiotic cells (e.g., brief formaldehyde followed by methanol)

• Co-localization studies:

  • Combine DYNLRB2 antibodies with markers for:
    a) Spindle microtubules (α/β-tubulin)
    b) Centrosomes (γ-tubulin, pericentrin)
    c) Nuclear Mitotic Apparatus protein (NuMA)
    d) Pericentriolar material components

• Analysis of meiotic stages:

  • Use DAPI staining to identify specific stages of meiosis

  • Correlate DYNLRB2 localization with spindle formation during metaphase I

  • Compare with Dynlrb2 knockout models showing multipolar spindles with fragmented PCM

• Advanced imaging:

  • Apply super-resolution microscopy for precise localization

  • Consider 3D reconstruction to visualize the entire meiotic spindle apparatus

Research has demonstrated that in Dynlrb2 KO mouse testes, meiosis progression is arrested in metaphase I due to the formation of multipolar spindles with fragmented pericentriolar material .

What are the technical considerations for detecting post-translational modifications of DYNLRB2?

Although specific post-translational modifications (PTMs) of DYNLRB2 are not extensively described in the provided search results, researchers investigating potential PTMs should consider these methodological approaches:

• Immunoprecipitation optimization:

  • Use highly specific DYNLRB2 antibodies for pull-down

  • Include phosphatase inhibitors and deacetylase inhibitors in lysis buffers

  • Consider crosslinking approaches for transient protein interactions

• Western blot analysis:

  • Employ Phos-tag gels for enhanced separation of phosphorylated forms

  • Use antibodies against common PTMs (phosphorylation, acetylation, ubiquitination)

  • Perform 2D gel electrophoresis to distinguish modified isoforms

• Mass spectrometry approaches:

  • Perform LC-MS/MS on immunoprecipitated DYNLRB2

  • Compare PTM profiles between different tissues or conditions

  • Focus on testis samples where DYNLRB2 is highly expressed

• Functional validation:

  • Generate site-directed mutants of predicted modification sites

  • Assess effects on spindle formation, dynein complex assembly, or viral trafficking

The observed molecular weight (11 kDa) being higher than the calculated weight (6 kDa) suggests potential post-translational modifications affecting DYNLRB2 migration during SDS-PAGE .

How can researchers investigate DYNLRB2's potential as a biomarker in hepatocellular carcinoma?

DYNLRB2 expression is significantly down-regulated in hepatocellular carcinoma (HCC) patients , suggesting potential as a diagnostic or prognostic biomarker. Researchers exploring this application should implement:

• Clinical sample analysis:

  • Perform immunohistochemistry on tissue microarrays containing:
    a) Normal liver tissue
    b) Cirrhotic liver tissue (pre-malignant)
    c) HCC samples of various grades and stages

  • Standardize staining protocols and scoring systems

• Correlation with clinical parameters:

  • Associate DYNLRB2 expression levels with:
    a) Tumor stage and grade
    b) Patient survival outcomes
    c) Response to specific therapies

  • Use multivariate analysis to assess independent prognostic value

• Mechanistic studies:

  • Investigate the functional consequences of DYNLRB2 downregulation in HCC

  • Examine effects on microtubule dynamics and mitotic spindle formation

  • Assess potential tumor suppressor functions

• Comparison with established HCC biomarkers:

  • Evaluate DYNLRB2 alongside AFP, GPC3, and other established markers

  • Determine if DYNLRB2 adds independent diagnostic or prognostic value

This research direction could provide insights into both the biological significance of DYNLRB2 in cancer progression and its potential clinical utility as a biomarker.

What are the best preservation methods for maintaining DYNLRB2 antibody quality?

To ensure optimal antibody performance in DYNLRB2 research, researchers should follow these evidence-based preservation protocols:

• Storage conditions:

  • Store antibodies at -20°C for long-term preservation

  • For short-term use (up to one month), store at 4°C

  • Avoid repeated freeze-thaw cycles that can damage antibody structure

  • Consider aliquoting stock solutions to minimize freeze-thaw cycles

• Buffer composition:

  • Most commercial DYNLRB2 antibodies are supplied in PBS containing:
    a) 50% glycerol as a cryoprotectant
    b) 0.02% sodium azide as a preservative
    c) 0.5% BSA for stability

  • Maintain these components when diluting stock solutions

• Quality control:

  • Include positive controls (mouse/rat testis tissue) with each experiment

  • Test antibody performance periodically against known standards

  • Document lot-to-lot variations when receiving new antibody stock

These preservation methods help maintain antibody binding affinity and specificity, ensuring reliable results in all experimental applications.

What controls are essential when studying DYNLRB2 in diverse experimental systems?

Rigorous control selection is critical for valid DYNLRB2 research across different experimental systems:

• Positive controls:

  • Mouse or rat testis tissue (high DYNLRB2 expression)

  • Cell lines with confirmed DYNLRB2 expression

  • Recombinant DYNLRB2 protein for antibody validation

• Negative controls:

  • DYNLRB2 knockdown/knockout models

  • Primary antibody omission

  • Isotype control antibodies

  • Tissues with naturally low DYNLRB2 expression

• Functional controls:

  • HA-tagged DYNLRB2 overexpression for rescue experiments

  • DYNLRB1 expression analysis for paralog comparison

  • Developmental stage controls (pre-pubertal vs. adult testis)

• Technical controls:

  • Loading controls for Western blots (β-actin, GAPDH)

  • Signal normalization references for immunofluorescence

  • Peptide competition assays to confirm binding specificity

These comprehensive controls enable confident interpretation of results and help distinguish specific DYNLRB2-related findings from experimental artifacts or background signals.

How might DYNLRB2 antibodies be utilized to explore novel therapeutic targets?

DYNLRB2's specialized roles suggest several promising therapeutic research avenues:

• Viral infection intervention:

  • Since DYNLRB2 is essential for murine leukemia virus traffic and nuclear localization , researchers could:
    a) Screen for small molecules that modulate DYNLRB2-viral interactions
    b) Develop peptide inhibitors targeting specific DYNLRB2 binding interfaces
    c) Investigate whether similar mechanisms apply to other retroviruses

• Male fertility applications:

  • Given DYNLRB2's critical role in meiotic spindle formation :
    a) Develop diagnostic tools for assessing DYNLRB2 expression in infertility cases
    b) Investigate DYNLRB2 mutations in patients with spermatogenesis defects
    c) Explore DYNLRB2-based contraceptive approaches

• Cancer therapeutic development:

  • Based on DYNLRB2's downregulation in hepatocellular carcinoma :
    a) Investigate whether restoring DYNLRB2 expression inhibits cancer progression
    b) Explore synthetic lethality approaches in DYNLRB2-deficient tumors
    c) Develop DYNLRB2-based diagnostic panels for early cancer detection

DYNLRB2 antibodies would be essential tools in these investigations for target validation, mechanism exploration, and therapeutic response assessment.

What emerging technologies could enhance DYNLRB2 antibody applications in research?

Several cutting-edge technologies could significantly advance DYNLRB2 research:

• Single-cell antibody-based technologies:

  • Single-cell proteomics to analyze DYNLRB2 expression heterogeneity

  • Mass cytometry (CyTOF) for high-dimensional analysis of DYNLRB2 and related proteins

  • Spatial transcriptomics combined with immunostaining to correlate protein expression with transcriptional profiles

• Advanced imaging approaches:

  • Live-cell super-resolution microscopy to track DYNLRB2 dynamics in real-time

  • Expansion microscopy for enhanced visualization of DYNLRB2 at centrosomes

  • Correlative light and electron microscopy (CLEM) to link DYNLRB2 localization with ultrastructural features

• Proximity-based methods:

  • BioID or TurboID approaches to identify proximal proteins in different cellular contexts

  • Proximity ligation assays to visualize DYNLRB2 interactions in situ

  • APEX2-based proximity labeling for identifying transient interaction partners

• Antibody engineering:

  • Development of recombinant antibody fragments with enhanced tissue penetration

  • Nanobody-based approaches for live-cell imaging of DYNLRB2

  • Bifunctional antibodies for targeted degradation of DYNLRB2-interacting proteins

These technologies could provide unprecedented insights into DYNLRB2's dynamic functions in different biological contexts.