PDS5B Antibody

What is PDS5B and what biological role does it play?

PDS5B functions as a regulator of sister chromatid cohesion in mitosis, working to stabilize cohesin complex association with chromatin. It plays a pivotal role in carcinogenesis and progression in certain cancer types . In non-small cell lung cancer, research demonstrates that PDS5B has tumor suppressor properties, as upregulation of PDS5B represses cell viability, migration, and invasion, while downregulation promotes these processes . PDS5B also appears to have tissue-specific functions, as its expression pattern differs from core cohesin components like SMC3 and STAG2, suggesting it modulates cohesin function in a tissue-specific manner .

What are the typical expression patterns of PDS5B in mammalian tissues?

Immunoblotting studies in mouse models reveal that PDS5B is highly expressed in specific tissues, with the highest protein expression levels found in testis and lung, followed by significant abundance in brain tissue . This tissue-specific expression pattern contrasts with that of core cohesin components (SMC3 and STAG2), which show much more uniform expression across tissues. This differential expression suggests PDS5B may have specialized regulatory functions in these highly expressing tissues . Understanding these tissue-specific expression patterns is crucial when selecting appropriate tissue controls for antibody validation.

What are the standard applications for PDS5B antibodies in research?

PDS5B antibodies are commonly utilized in several standard molecular and cellular biology techniques:

The rabbit polyclonal antibodies to PDS5B are typically reactive against human, mouse, and rat samples, with predicted cross-reactivity to zebrafish, bovine, horse, sheep, rabbit, and dog samples .

What is the molecular weight of PDS5B protein?

The molecular weight of PDS5B protein is approximately 165 kDa (calculated) . This information is essential when verifying the specificity of antibody binding in western blot applications, where researchers should expect to see a band at approximately this position. Variations in observed molecular weight may occur due to post-translational modifications or alternative splicing variants of the protein.

How should I optimize Western blot protocols for detecting PDS5B in different tissue samples?

For optimal detection of PDS5B via Western blotting, consider the following methodological approach:

• Sample preparation: Prepare nuclear extracts as these provide better enrichment of PDS5B. Homogenize tissues in a buffer containing 0.32 M Sucrose, 10 mM HEPES pH 7.4, 1 mM PMSF, and complete protease inhibitor cocktail .

• Nuclear extraction: After centrifugation at 1,000 g, resuspend the pellet in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, and protease inhibitors) .

• Protein separation: Use lower percentage gels (6-8%) to effectively resolve the 165 kDa PDS5B protein.

• Transfer conditions: For large proteins like PDS5B, consider extended transfer times or higher voltage transfers to PVDF membranes.

• Blocking and antibody conditions: Use 5% non-fat milk or BSA in TBST. Primary anti-PDS5B antibody dilution of 1:1,000 has been reported effective .

• Positive controls: Include testis or lung tissue extracts as positive controls due to high endogenous expression .

• Validation: Confirm specificity using multiple antibodies against different epitopes of PDS5B, as performed in previous studies using different anti-PDS5B antibodies (e.g., Cat#IHC-00381 and Cat#A300-538A from Bethyl Laboratories) .

What are the recommended approaches for studying PDS5B interactions with the cohesin complex?

To effectively study PDS5B interactions with the cohesin complex, implement these methodological strategies:

• Co-immunoprecipitation (Co-IP): Homogenize tissues (preferably testis due to high expression) in 0.32 M Sucrose buffer with 10 mM HEPES pH 7.4, 1 mM PMSF, and protease inhibitors. After initial centrifugation, resuspend the pellet in IP buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS, and protease inhibitors) . Use antibodies against PDS5B or other cohesin components (SMC1β, SYCP2, HORMAD1, REC8) for immunoprecipitation.

• Proximity ligation assays (PLA): This technique can visualize protein-protein interactions in situ, allowing for detection of PDS5B interactions with cohesin components in their native cellular context.

• ChIP-seq: To investigate genomic binding sites of PDS5B in relation to cohesin complex components, especially at loop anchors where PDS5B is known to stabilize cohesin .

• Nuclear spreads with co-immunofluorescence: Prepare nuclear spreads according to established protocols for surface spreading of meiotic chromosomes . Use anti-PDS5B antibody (1:50 dilution) alongside antibodies against cohesin components like SYCP3 (1:400) and REC8 (1:100) to visualize co-localization.

How can I troubleshoot non-specific binding when using PDS5B antibodies?

When encountering non-specific binding with PDS5B antibodies, implement these troubleshooting strategies:

• Peptide competition assay: Confirm specificity by pre-incubating the antibody with the peptide corresponding to the epitope. This approach has been successfully used to validate PDS5B staining specificity in previous studies .

• Multiple antibody validation: Use alternative antibodies targeting different epitopes of PDS5B to confirm consistent binding patterns. Previous research confirmed specificity by comparing results with two different anti-PDS5B antibodies (Cat#IHC-00381 and Cat#A300-538A from Bethyl Laboratories) .

• Optimize blocking conditions: Test different blocking reagents (BSA, non-fat milk, commercial blockers) and concentrations to reduce background.

• Antibody dilution optimization: Test a range of antibody dilutions to find the optimal signal-to-noise ratio. Published studies have used 1:1,000 for Western blotting and 1:50 for immunofluorescence .

• Sample preparation: Ensure complete denaturation for Western blotting. For nuclear proteins like PDS5B, proper nuclear extraction protocols are critical.

• Knockout/knockdown controls: When available, use samples from PDS5B knockout or knockdown models as negative controls.

• Cross-reactivity assessment: Test the antibody on samples from species not expected to cross-react with the antibody to identify potential cross-reactivity issues.

What methodologies are most effective for studying PDS5B's role in chromatin loop formation?

To investigate PDS5B's role in chromatin loop formation, the following methodological approaches are recommended:

• Hi-C and derivatives: These chromosome conformation capture techniques can identify chromatin loops at the genome-wide level. Compare wild-type and PDS5B-depleted cells to determine how PDS5B affects loop formation and maintenance .

• ChIP-seq: Perform chromatin immunoprecipitation followed by sequencing to map PDS5B binding sites across the genome, particularly at loop anchors. Co-binding analysis with other cohesin components can reveal their spatial relationships .

• Live-cell imaging: Use fluorescently tagged PDS5B and other cohesin components to track their dynamics during loop formation in real-time.

• Genome editing: Use CRISPR-Cas9 to create specific mutations in PDS5B and assess effects on loop formation through Hi-C or microscopy approaches.

• Acute protein degradation systems: Employ auxin-inducible degradation (AID) or similar systems to acutely degrade PDS5B proteins and observe immediate effects on chromosome architecture . This approach helps distinguish direct effects from compensatory mechanisms.

• Immunofluorescence with 3D preservation: Use super-resolution microscopy techniques after immunofluorescence staining to visualize the spatial organization of chromosomal loops and PDS5B localization.

Research indicates that PDS5 proteins stabilize cohesin at loop anchors, facilitate chromatin loop formation, and restrict loop expansion in mammals , making these approaches particularly relevant for understanding its mechanistic role.

How do I interpret changes in PDS5B expression levels in cancer research?

When interpreting PDS5B expression data in cancer research, consider these important factors:

• Tumor suppressor role: Studies show that PDS5B upregulation represses cell viability, migration, and invasion in non-small cell lung cancer (NSCLC) cells, while downregulation promotes these processes . Therefore, decreased PDS5B expression may indicate more aggressive cancer phenotypes.

• Clinical correlation: Low expression of PDS5B has been associated with lymph node metastasis in lung cancer patients , suggesting its potential use as a prognostic biomarker.

• Mechanistic relationships: PDS5B positively regulates LATS1 expression in NSCLC cells , indicating its involvement in tumor suppressor pathways. When analyzing expression data, consider downstream effectors and related pathway components.

• In vivo validation: PDS5B overexpression retards tumor growth in nude mice , supporting in vitro findings. Compare tissue culture and animal model data for consistency.

• Tissue specificity: PDS5B shows tissue-specific expression patterns , so expression changes should be interpreted relative to appropriate control tissues.

• Quantification methods: When comparing studies, consider differences in quantification methods (immunohistochemistry, western blotting, RT-PCR) which may yield different results.

• Isoform analysis: Check whether the analysis distinguishes between potential isoforms of PDS5B, as different isoforms may have distinct functions.

What are the critical controls needed for validating PDS5B antibody specificity?

To properly validate PDS5B antibody specificity, implement these critical controls:

• Positive tissue controls: Include tissues known to have high PDS5B expression (testis, lung, brain) based on previous characterization .

• Negative controls: Use tissues with minimal PDS5B expression or samples from knockout/knockdown models when available.

• Peptide competition assay: Pre-incubate the antibody with the peptide corresponding to the epitope to confirm binding specificity. This approach has been used successfully to validate PDS5B staining specificity in previous studies .

• Multiple antibody validation: Use different antibodies targeting distinct epitopes of PDS5B. Previous research confirmed specificity by comparing results with two different anti-PDS5B antibodies (Cat#IHC-00381 and Cat#A300-538A from Bethyl Laboratories) .

• Expected molecular weight verification: Confirm detection at the expected molecular weight (165 kDa) in Western blot applications .

• Cross-reactivity assessment: Test the antibody on samples from species not predicted to cross-react with the antibody to identify potential cross-reactivity issues.

• Subcellular localization confirmation: Verify that the antibody detects PDS5B in the expected nuclear localization, consistent with its role in chromatin regulation.

• siRNA/shRNA knockdown: Demonstrate reduced antibody signal following PDS5B knockdown, as shown in previous studies where siRNA transfection effectively downregulated PDS5B expression .

How should I analyze PDS5B localization during different stages of the cell cycle?

For analyzing PDS5B localization across cell cycle stages, implement these methodological steps:

• Synchronization: Use established cell synchronization methods (double thymidine block, nocodazole treatment, or mitotic shake-off) to obtain populations enriched in specific cell cycle phases.

• Co-staining approaches: Combine PDS5B antibody staining with markers for specific cell cycle phases:

  • G1 phase: Cyclin D

  • S phase: PCNA, EdU incorporation, or BrdU pulse-labeling

  • G2 phase: Cyclin B (cytoplasmic)

  • Mitosis: Phospho-histone H3, Cyclin B (nuclear)

• Live-cell imaging: For dynamic changes, use fluorescently tagged PDS5B in live-cell imaging with cell cycle markers.

• Chromosome spreads: For meiotic cells, prepare nuclear spreads according to established protocols for surface spreading . Use anti-PDS5B antibody (1:50 dilution) alongside meiotic stage-specific markers.

• Super-resolution microscopy: Employ techniques like structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) to precisely localize PDS5B relative to chromosomal structures.

• Chromatin fractionation: Biochemically separate chromatin-bound and soluble nuclear fractions across synchronized cell populations to quantify PDS5B association with chromatin during different cell cycle phases.

• Quantitative analysis: Develop algorithms to quantify signal intensity, co-localization coefficients, and spatial distribution patterns of PDS5B throughout the cell cycle.

• Statistical validation: Analyze multiple cells (n>100) per condition to account for cell-to-cell variability and ensure results are statistically significant.

What is the optimal protocol for PDS5B immunoprecipitation from nuclear extracts?

For optimal PDS5B immunoprecipitation from nuclear extracts, follow this detailed protocol:

• Tissue/cell preparation:

  • For tissues (preferably testis due to high expression), homogenize in buffer containing 0.32 M Sucrose, 10 mM HEPES pH 7.4, 1 mM PMSF, and complete protease inhibitor cocktail

  • Centrifuge at 1,000 g to pellet nuclei

• Nuclear extraction:

  • Resuspend the nuclear pellet in IP buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS, and protease inhibitors)

  • Centrifuge at 16,000 g

  • Resuspend pellet in IP buffer, sonicate, and centrifuge again at 16,000 g

• Pre-clearing:

  • Pre-clear the supernatant with protein A-conjugated Dynabeads to reduce non-specific binding

• Immunoprecipitation:

  • Incubate pre-cleared lysate with anti-PDS5B antibody bound to protein A-conjugated Dynabeads

  • Anti-PDS5B antibodies from Bethyl Laboratories have been successfully used for this purpose

  • Include control IPs with antibodies against known interacting partners (SYCP2, HORMAD1, REC8, SMC1β) for validation

• Washing:

  • Wash immunoprecipitates extensively in RIPA buffer to reduce background

• Elution:

  • Elute bound proteins in LDS protein sample buffer

• Analysis:

  • Subject eluted proteins to SDS-PAGE and immunoblot analysis with antibodies against PDS5B and potential interacting partners

  • Consider silver staining and mass spectrometry for unbiased identification of novel interacting partners

This protocol has been validated in published studies examining PDS5B interactions in testicular cells .

How can I design experiments to study PDS5B's tumor suppressor function?

To investigate PDS5B's tumor suppressor function, implement these experimental approaches:

• Expression modulation studies:

  • Overexpression: Transfect PDS5B cDNA into cancer cell lines and assess effects on proliferation, migration, invasion, and apoptosis

  • Knockdown: Use siRNA or shRNA to downregulate PDS5B expression and evaluate the same parameters

• Cell-based functional assays:

  • Cell viability: MTT or CCK-8 assays following PDS5B modulation

  • Migration: Wound-healing assays and transwell migration assays

  • Invasion: Transwell invasion assays with Matrigel coating

  • Colony formation: Assess long-term proliferative capacity

• Molecular mechanism investigation:

  • Assess effects on downstream pathways (e.g., LATS1 expression which is positively regulated by PDS5B in NSCLC cells)

  • Perform RNA-seq or proteomics analysis after PDS5B modulation to identify affected pathways

  • Conduct chromatin immunoprecipitation to identify genomic targets

• In vivo validation:

  • Xenograft models: Implant cells with modified PDS5B expression in nude mice and monitor tumor growth

  • Measure tumor weights and volumes, as shown effective in previous studies

  • Analyze tumor tissues for PDS5B and downstream effector expression

• Clinical correlation:

  • Analyze PDS5B expression in patient samples and correlate with clinical parameters

  • Previous studies found association between low PDS5B expression and lymph node metastasis in lung cancer patients

• Combinatorial approaches:

  • Test PDS5B expression modulation in combination with standard chemotherapies

  • Explore synthetic lethality with other genetic alterations

These approaches have demonstrated that PDS5B functions as a tumor suppressor in non-small cell lung cancer, with its upregulation inhibiting proliferation, migration, and invasion both in vitro and in vivo .

What methods are most suitable for studying PDS5B in meiotic versus mitotic cells?

For comparative analysis of PDS5B in meiotic versus mitotic cells, employ these specialized methods:

• Tissue selection:

  • Meiotic cells: Testis tissue provides an excellent source of meiotic cells at various stages

  • Mitotic cells: Proliferating somatic tissues or cultured cell lines

  • Comparative analysis: The contrasting expression patterns make this comparison particularly informative

• Chromosome spreads for meiotic cells:

  • Follow established protocols for surface spreading of meiotic chromosomes

  • Use anti-PDS5B antibody (1:50 dilution) with meiotic markers like SYCP3 (1:400) and REC8 (1:100)

  • Visualize using fluorescence microscopy with appropriate secondary antibodies

• Staging of meiotic cells:

  • Use stage-specific markers to identify different phases of meiotic prophase I

  • Co-stain with SYCP3 to visualize synaptonemal complex formation and identify prophase stages

  • Analyze PDS5B localization patterns specific to each meiotic stage

• Mitotic cell analysis:

  • Synchronize cells in different mitotic phases

  • Use mitotic markers like phospho-histone H3

  • Perform immunofluorescence to visualize PDS5B localization during mitosis

• Protein complex analysis:

  • Compare immunoprecipitation results between meiotic and mitotic cells

  • Identify cell type-specific interaction partners

  • Analyze differential association with cohesin components

• Genetic models:

  • Utilize mouse models with mutations in meiosis-specific genes (Sycp1-/-, Smc1β-/-, and Sycp3-/-) to understand PDS5B dependency on these factors

  • Compare effects of PDS5B depletion in meiotic versus mitotic contexts

• Quantitative analysis:

  • Develop algorithms to quantify localization patterns specific to meiotic versus mitotic chromosomes

  • Measure co-localization coefficients with various markers

This comparative approach leverages the differential expression and function of PDS5B in reproductive versus somatic tissues, providing insights into its specialized roles in these distinct cell division processes .

How should I optimize ELISA protocols for PDS5B detection?

For optimizing PDS5B ELISA detection, implement these technical considerations based on established protocols:

• Assay principle selection:

  • Competitive ELISA is recommended for PDS5B detection, utilizing a monoclonal anti-PDS5B antibody and a PDS5B-HRP conjugate

  • In this format, PDS5B from samples competes with PDS5B-HRP conjugate for binding to the antibody-coated plate

• Protocol optimization:

  • Incubate samples and buffer with PDS5B-HRP conjugate in pre-coated plates for one hour

  • After incubation, decant and wash wells five times

  • Add substrate for HRP enzyme, resulting in a blue colored complex

  • Add stop solution to turn the solution yellow

  • Measure intensity spectrophotometrically at 450nm

• Standard curve preparation:

  • Create a dilution series of purified PDS5B protein

  • Plot the relationship between optical density and concentration

  • The intensity of color is inversely proportional to PDS5B concentration in the sample

• Sample preparation:

  • For tissue homogenates, use appropriate extraction buffers with protease inhibitors

  • Consider using undiluted body fluids and/or tissue homogenates, secretions as recommended

  • Centrifuge samples to remove particulates before testing

• Quality control measures:

  • Include intra-assay and inter-assay controls to assess reproducibility

  • Calculate coefficient of variation (CV) for both parameters

  • Include positive and negative controls in each run

• Sensitivity and specificity validation:

  • Determine the lower limit of detection and quantification

  • Assess cross-reactivity with related proteins

  • Validate results against other methods (Western blot, mass spectrometry)

• Troubleshooting:

  • High background: Increase washing steps or optimize blocking

  • Poor standard curve: Check reagent quality and preparation

  • Low signal: Optimize antibody concentrations or incubation times

This approach is based on established ELISA protocols for PDS5B detection that have demonstrated effective quantification of this protein in research applications .

What are the key considerations when designing PDS5B knockdown or knockout experiments?

When designing PDS5B knockdown or knockout experiments, consider these critical factors:

• Method selection based on experimental goals:

  • Transient knockdown (siRNA): Ideal for short-term effects analysis as demonstrated in previous NSCLC studies

  • Stable knockdown (shRNA): Better for long-term studies and in vivo experiments

  • CRISPR-Cas9 knockout: For complete elimination of protein expression

  • Conditional knockout: For tissue-specific or temporal control, especially important given PDS5B's tissue-specific expression patterns

• Validation strategies:

  • Confirm knockdown/knockout efficiency at both mRNA (qRT-PCR) and protein (Western blot) levels

  • Previous studies successfully validated PDS5B knockdown in H1975 and H460 NSCLC cell lines

  • Use multiple siRNA sequences or guide RNAs targeting different regions to minimize off-target effects

• Functional readouts:

  • Cell viability assays (MTT) as performed in NSCLC studies

  • Migration assays (wound-healing and transwell migration)

  • Invasion assays (Matrigel-coated transwell)

  • Chromosome cohesion analysis

  • Cell cycle progression assessment

• Control selection:

  • Non-targeting siRNA/shRNA controls

  • Empty vector controls for overexpression studies

  • Wild-type cells for CRISPR experiments

  • Rescue experiments by reintroducing PDS5B to confirm phenotype specificity

• Temporal considerations:

  • Monitor phenotypes at multiple time points post-knockdown/knockout

  • Consider potential compensatory mechanisms (e.g., PDS5A upregulation)

• In vivo validation:

  • Previous studies successfully used nude mice xenograft models with PDS5B-modulated cells

  • Monitor tumor growth, weight, and volume over time

  • Analyze tissue samples for marker expression

• Mechanistic investigations:

  • Assess effects on known pathways (e.g., LATS1 expression)

  • Examine effects on chromosome cohesion and chromatin loop formation

  • Perform "-omics" analyses to identify global changes

• Physiological relevance:

  • Consider tissue-specific effects, particularly in testis, brain, and lung where PDS5B is highly expressed

  • Relate findings to relevant disease contexts like cancer