PSMD13 Antibody

What types of PSMD13 antibodies are available for research applications?

Research laboratories have access to several types of PSMD13 antibodies, primarily including rabbit polyclonal antibodies and rabbit recombinant monoclonal antibodies. Polyclonal antibodies recognize multiple epitopes on PSMD13, offering high sensitivity but potentially with more background noise . Monoclonal antibodies, particularly recombinant versions like EPR8524, recognize a single epitope, providing superior specificity and consistency . These antibodies are available in various formats, including unconjugated forms in buffered aqueous glycerol solutions that maintain stability during storage . The choice between polyclonal and monoclonal depends on the specific experimental requirements, with each offering distinct advantages for different applications.

What applications are PSMD13 antibodies validated for?

PSMD13 antibodies have been validated for multiple research applications. Western blotting (WB) is widely supported, with recommended dilutions typically ranging from 1:500-1:2000 . Immunohistochemistry on paraffin-embedded tissues (IHC-P) is another common application, with dilutions of 1:50-1:500 . Immunofluorescence and immunocytochemistry (IF/ICC) applications generally use dilutions of 1:10-1:100 or 0.25-2 μg/mL . Immunoprecipitation (IP) is also supported by certain antibodies, with recommendations of 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate . Some antibodies are additionally validated for ELISA applications . Researchers should review specific product documentation for optimal conditions in their experimental systems.

What is the expected molecular weight of PSMD13 in Western blot analysis?

The expected molecular weight of PSMD13 in Western blot analysis is consistently reported as 43 kDa . This has been confirmed across multiple antibodies and cell lines, including HEK-293, HepG2, HeLa, Jurkat, and Y79 cells . Both the calculated molecular weight based on amino acid sequence and the observed molecular weight in experimental conditions align at 43 kDa, indicating that PSMD13 typically runs true to its predicted size on SDS-PAGE gels. This consistent band size provides a reliable indicator for identifying PSMD13 in Western blot applications.

What species reactivity do commercial PSMD13 antibodies demonstrate?

Most commercial PSMD13 antibodies demonstrate strong reactivity with human samples, as consistently reported across manufacturers . Some antibodies also show cross-reactivity with mouse and rat samples due to sequence homology across these species . For example, the HPA038692 antibody specifically notes reactivity with human, mouse, and rat samples . Researchers should carefully verify the species reactivity claims for their specific antibody of interest, noting whether the reactivity has been experimentally validated or is predicted based on sequence homology. This information is crucial for experimental design, particularly in comparative studies using animal models.

How should Western blot conditions be optimized for PSMD13 detection?

Optimizing Western blot conditions for PSMD13 detection requires attention to several key parameters. For protein loading, 10 μg of total protein per lane is commonly used with success in cell lysates from HEK-293, HepG2, HeLa, Jurkat, and Y79 cells . Antibody dilutions typically range from 1:500-1:2000 for primary antibodies, with incubation preferably overnight at 4°C for optimal signal development . Secondary antibody selection should match the host species (typically anti-rabbit for most PSMD13 antibodies), with HRP-conjugated antibodies commonly used at 1:2000-1:10000 dilutions . For membrane transfer, standard PVDF membranes are suitable for the 43 kDa PSMD13 protein. Given the moderate molecular weight of PSMD13, standard 10-12% polyacrylamide gels provide adequate separation. Blocking with 5% non-fat milk or BSA in TBST for 1 hour at room temperature is typically sufficient before antibody application.

What are the recommended protocols for immunohistochemistry with PSMD13 antibodies?

For successful immunohistochemistry with PSMD13 antibodies, proper tissue preparation and antigen retrieval are critical. Formalin-fixed, paraffin-embedded tissues are commonly used, with sections typically cut at 4-5 μm thickness. Antigen retrieval methods vary, with TE buffer (pH 9.0) being the primary recommendation, though citrate buffer (pH 6.0) is an acceptable alternative . Following deparaffinization and antigen retrieval, sections should undergo endogenous peroxidase blocking (typically 3% H₂O₂ for 10 minutes) and protein blocking (5-10% normal serum for 30-60 minutes). PSMD13 antibodies are typically applied at dilutions ranging from 1:50-1:500, with incubation times of 1-2 hours at room temperature or overnight at 4°C . Detection systems using HRP-conjugated secondary antibodies with DAB substrate provide reliable visualization. This protocol has been validated on multiple tissue types, including lung cancer, adrenal gland, and colon tissues .

How can researchers validate the specificity of PSMD13 antibodies in experimental systems?

Validating the specificity of PSMD13 antibodies requires a multi-faceted approach. Positive controls should include cell lines known to express PSMD13, such as HEK-293, HeLa, HepG2, Jurkat, or Y79 cells as referenced in the product literature . Negative controls should ideally include PSMD13 knockdown or knockout samples to confirm signal specificity. Western blot analysis should verify the presence of a single band at the expected 43 kDa molecular weight . For immunohistochemistry or immunofluorescence applications, comparing staining patterns to known PSMD13 subcellular localization provides another validation criterion. Cross-validation using multiple antibodies targeting different epitopes of PSMD13 can further confirm specificity. Some manufacturers employ enhanced validation techniques, including independent verification methods, which provide additional confidence in antibody specificity . Researchers should document their validation process thoroughly, including specific positive and negative controls used.

What controls are essential when working with PSMD13 antibodies?

A comprehensive experimental design with PSMD13 antibodies requires several types of controls. Positive sample controls should include cell lines with confirmed PSMD13 expression, such as HEK-293, HeLa, HepG2, Jurkat, or Y79 cells . Technical negative controls should include omission of primary antibody while maintaining all other steps to identify non-specific binding from secondary antibodies. For quantitative applications, loading controls such as GAPDH, β-actin, or total protein staining are essential to normalize PSMD13 expression data. When possible, biological negative controls using PSMD13 knockdown/knockout samples provide the strongest evidence of antibody specificity. For immunohistochemistry applications, including irrelevant tissues known not to express PSMD13 can help establish staining specificity. Additionally, using isotype control antibodies matched to the primary antibody class and concentration can identify non-specific binding related to the antibody class rather than antigen specificity.

How do monoclonal and polyclonal PSMD13 antibodies perform in different applications?

The performance of monoclonal versus polyclonal PSMD13 antibodies varies by application context. Monoclonal antibodies like EPR8524 offer superior specificity and consistency, particularly in Western blot applications where clean, single-band detection is critical . These antibodies typically produce lower background and more reproducible results across different experiments and antibody lots. Polyclonal antibodies provide advantages in sensitivity, as they recognize multiple epitopes on PSMD13, making them valuable for detecting low-abundance protein or partially denatured proteins in fixed tissues . For immunohistochemistry applications, polyclonal antibodies sometimes provide stronger signal in heavily fixed tissues due to their multi-epitope recognition. For immunoprecipitation studies, both types can be effective, though monoclonal antibodies may offer cleaner results with fewer contaminating proteins. The choice between these antibody types should be guided by the specific application requirements, with consideration of the relative importance of sensitivity versus specificity.

How can PSMD13 antibodies be employed to study proteasome dysfunction in disease models?

PSMD13 antibodies serve as valuable tools for investigating proteasome dysfunction in various disease contexts. In neurodegenerative disease models, these antibodies can be used in immunohistochemistry to assess alterations in PSMD13 expression patterns or subcellular localization in affected brain regions. Western blot analysis using PSMD13 antibodies can quantify changes in protein levels across disease progression stages or in response to therapeutic interventions . Co-immunoprecipitation experiments with PSMD13 antibodies can reveal altered interactions with other proteasome components or disease-specific proteins. Immunofluorescence microscopy can visualize co-localization of PSMD13 with pathological protein aggregates. For in vitro studies, PSMD13 antibodies can monitor proteasome integrity following exposure to stressors or potential therapeutic compounds. When interpreting results, researchers should consider that changes in PSMD13 levels may represent compensatory responses rather than primary disease mechanisms, necessitating careful experimental design with appropriate controls.

What protocols are recommended for co-immunoprecipitation with PSMD13 antibodies?

For successful co-immunoprecipitation (co-IP) studies with PSMD13 antibodies, researchers should implement the following protocol: Begin with cell lysis under non-denaturing conditions using buffers containing mild detergents (0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions. Pre-clear lysates with protein A/G beads to reduce non-specific binding. For immunoprecipitation, use 0.5-4.0 μg of PSMD13 antibody per 1.0-3.0 mg of total protein lysate, as recommended in the product documentation . Incubate with gentle rotation overnight at 4°C. Add pre-washed protein A/G beads and continue incubation for 1-2 hours. Perform stringent washing steps with decreasing detergent concentrations. Elute immunoprecipitated complexes using denaturing buffer for subsequent SDS-PAGE analysis. For detection, use antibodies against other proteasome components or interaction partners of interest. This approach has been validated in HEK-293T cells and can be adapted for studying how various conditions affect proteasome complex assembly or PSMD13 interactions .

How can researchers use PSMD13 antibodies to investigate proteasome regulation in cancer progression?

Investigating proteasome regulation in cancer progression using PSMD13 antibodies requires a multi-faceted experimental approach. Immunohistochemistry with PSMD13 antibodies can assess expression patterns across tumor grades, stages, and subtypes, potentially identifying correlations with clinical outcomes . Tissue microarray analysis allows high-throughput screening across multiple patient samples. Western blot analysis of cancer cell lines with varying metastatic potential can establish relationships between PSMD13 levels and malignant phenotypes . Functional studies combining PSMD13 knockdown or overexpression with antibody-based detection can determine causality in cancer-related processes. Co-immunoprecipitation experiments can identify cancer-specific interaction partners. For mechanistic studies, immunofluorescence microscopy can track PSMD13 redistribution in response to chemotherapeutic agents or targeted proteasome inhibitors. When designed with appropriate controls, these approaches can provide valuable insights into how proteasome function contributes to cancer progression and therapy response.

What approaches can be used to study the spatial distribution of PSMD13 in cellular compartments?

Studying the spatial distribution of PSMD13 requires complementary techniques leveraging specific antibodies. Immunofluorescence microscopy provides direct visualization of PSMD13 localization patterns, with recommended antibody dilutions of 1:10-1:100 . Counter-staining with organelle markers (DAPI for nuclei, MitoTracker for mitochondria, etc.) enables precise localization. For higher resolution analysis, super-resolution microscopy (STORM, STED) can be employed with appropriate fluorophore-conjugated secondary antibodies. Cell fractionation followed by Western blotting with PSMD13 antibodies allows biochemical quantification of distribution across subcellular compartments. Proximity ligation assays combining PSMD13 antibodies with antibodies against compartment-specific proteins can detect in situ associations. Immunoelectron microscopy provides ultrastructural localization information. For analysis in tissue context, immunohistochemistry at dilutions of 1:50-1:500 reveals cell-type specific distribution patterns . These complementary approaches provide a comprehensive view of PSMD13 distribution across cellular compartments in normal and pathological conditions.

How do post-translational modifications of PSMD13 affect antibody recognition?

Post-translational modifications (PTMs) of PSMD13 can significantly impact antibody recognition and experimental outcomes. When using PSMD13 antibodies, researchers should consider the immunogen sequence against which the antibody was raised. For example, some antibodies target specific sequences like LEKTREKVKSSDEAVILCKTAIGALKLNIGDLQVTKETIEDVEEMLNNLPGVTSVHSRFYDLSSKYYQTIGNHASYYKDALRFLGCVDIKDLPVSEQQE , while others target recombinant fragments within the first 250 amino acids . If PTMs occur within these regions, antibody binding may be enhanced or inhibited. To address this complexity, researchers can employ phosphatase or deubiquitinase treatments before immunodetection to remove specific modifications. Two-dimensional gel electrophoresis followed by Western blotting can separate PSMD13 isoforms based on charge differences caused by PTMs. When unexpected band patterns emerge in Western blots, researchers should consider whether these might represent differently modified forms of PSMD13 rather than non-specific binding.

What causes multiple bands in Western blots with PSMD13 antibodies?

Multiple bands in Western blots using PSMD13 antibodies may arise from several sources that researchers should systematically evaluate. Alternative splicing of the PSMD13 gene could generate isoforms with different molecular weights. Post-translational modifications like phosphorylation or ubiquitination can alter protein mobility, creating additional bands. Proteolytic degradation during sample preparation may produce fragments recognized by the antibody—using fresh samples with protease inhibitors and maintaining samples at 4°C can minimize this issue. Non-specific binding, particularly with polyclonal antibodies, may occur—optimizing blocking and washing conditions can reduce this problem. The expected molecular weight of PSMD13 is 43 kDa , which should serve as the reference point when interpreting bands. To determine which band represents true PSMD13, validation with PSMD13 knockdown/knockout samples provides definitive evidence. Testing multiple antibodies targeting different epitopes can also help distinguish specific from non-specific signals.

How can researchers address non-specific background in immunohistochemistry with PSMD13 antibodies?

Addressing non-specific background in immunohistochemistry with PSMD13 antibodies requires systematic optimization. First, titrate antibody concentration within the recommended range of 1:50-1:500 to identify the optimal dilution that provides specific signal with minimal background. Enhance blocking steps by extending time (1-2 hours) or increasing blocking reagent concentration (5-10% normal serum). Optimize antigen retrieval conditions, testing both recommended methods: TE buffer at pH 9.0 and citrate buffer at pH 6.0 . Extend washing steps between incubations, using at least three 5-minute washes with fresh buffer. Consider using more specific detection systems, such as polymer-based detection rather than avidin-biotin methods to reduce endogenous biotin-related background. Include appropriate controls—sections without primary antibody and isotype controls help distinguish specific from non-specific staining. For challenging tissues with high background, consider using amplification systems with more stringent washing or treating sections with background-reducing reagents before antibody application.

What factors contribute to variability in PSMD13 detection across different cell lines?

Variability in PSMD13 detection across cell lines stems from both biological and technical factors. Biological differences in PSMD13 expression levels naturally exist between cell types—published data confirms detection in HEK-293, HepG2, HeLa, Jurkat, and Y79 cells, but with varying signal intensities . Cell-type specific post-translational modifications may affect antibody recognition. Protein extraction efficiency varies based on cell characteristics—optimizing lysis buffers (detergent concentration, mechanical disruption methods) for specific cell types improves consistency. Culture conditions (confluence, passage number, media composition) can alter proteasome composition and PSMD13 expression—standardization is essential for reproducible results. To address variability, researchers should validate antibodies in each cell line of interest, establish standardized protocols for sample preparation, use loading controls appropriate for the cell types being compared, and if possible, employ multiple PSMD13 antibodies targeting different epitopes to confirm findings.

How should contradictory results between different PSMD13 antibodies be interpreted?

Interpreting contradictory results between different PSMD13 antibodies requires systematic analysis. First, examine epitope differences—antibodies targeting different regions of PSMD13 may perform differently depending on protein conformation or modifications. For example, some antibodies target recombinant fragments within the first 250 amino acids , while others target other regions. Second, consider format differences—monoclonal antibodies like EPR8524 provide high specificity but might miss certain protein variants, while polyclonal antibodies offer broader detection but potentially more background. Third, acknowledge application-specific performance—some antibodies excel in Western blot but perform poorly in IHC. Fourth, validate with orthogonal methods—confirm protein expression using RNA analysis or mass spectrometry. When reporting contradictory results, document the exact antibody used (catalog number, lot, dilution), experimental conditions, and controls. Consider that both results might be correct but reflecting different aspects of PSMD13 biology, such as different isoforms or post-translationally modified forms.

What strategies can resolve loss of signal in experiments using PSMD13 antibodies?

Signal loss in experiments using PSMD13 antibodies can be addressed through systematic troubleshooting. For antibody-related issues, verify proper storage conditions (typically -20°C with 50% glycerol as indicated in product documentation ) and avoid repeated freeze-thaw cycles. For antigen accessibility problems, optimize antigen retrieval methods for fixed tissues, testing both recommended approaches (TE buffer pH 9.0 and citrate buffer pH 6.0 ). In Western blot applications, insufficient protein transfer or protein degradation may cause signal loss—optimize transfer conditions for the 43 kDa PSMD13 protein and include protease inhibitors during sample preparation. If signal loss occurs in specific samples but not controls, consider whether biological conditions might be downregulating PSMD13 expression. For low-abundance detection, employ signal amplification methods such as HRP-polymer detection systems or more sensitive chemiluminescent substrates. If these approaches fail, testing alternative PSMD13 antibodies targeting different epitopes may resolve the issue, as different regions of the protein may be more accessible in specific experimental contexts.

Recommended Antibody Dilutions for Different Applications

Validated Cell Lines for PSMD13 Expression Detection