PSMD2 Antibody

What is PSMD2 and what is its significance in cellular function?

PSMD2 (proteasome 26S subunit, non-ATPase 2) is a critical component of the 26S proteasome, which is responsible for ATP-dependent degradation of ubiquitinated proteins. This complex plays an essential role in maintaining protein homeostasis by removing misfolded or damaged proteins that could impair cellular functions, as well as removing proteins whose functions are no longer required . PSMD2 is also known by several alternative names including TRAP2, 26S proteasome regulatory subunit RPN1, 26S proteasome regulatory subunit S2, and 26S proteasome subunit p97 . As a component of this multiprotein complex, PSMD2 participates in numerous cellular processes, including cell cycle progression, apoptosis, and DNA damage repair . Interestingly, it also binds to the intracellular domain of tumor necrosis factor type 1 receptor, with the binding domain residing outside the death domain of TNFR1 . This multifunctional nature of PSMD2 makes it an important target for investigating proteasome function in various biological processes.

How can researchers distinguish between different types of PSMD2 antibodies available for research?

Researchers should evaluate PSMD2 antibodies based on multiple criteria including host species, clonality, epitope recognition, and validated applications. Polyclonal antibodies, such as the rabbit polyclonal (14748-1-AP) or goat polyclonal (ab26078) options, typically recognize multiple epitopes of PSMD2 and offer high sensitivity but may have batch-to-batch variation . When selecting between antibodies, researchers should verify:

• Epitope location: For example, ab26078 recognizes an epitope within Human PSMD2 aa 850 to C-terminus, while other antibodies may target different regions .

• Validated applications: Some antibodies are validated for multiple applications (WB, IHC, IF, IP, CoIP, ELISA) while others have more limited validated uses .

• Reactivity with species of interest: Confirm the antibody reacts with your experimental model. Most PSMD2 antibodies show reactivity with human, mouse, and rat samples, but predicted reactivity can extend to other species like pig, bovine, and sheep .

The choice between antibodies should be guided by the specific experimental requirements and the technical validation data provided by manufacturers.

What is the molecular weight of PSMD2 and how does this impact antibody detection methods?

PSMD2 has a calculated molecular weight of 100 kDa, which is consistent with the observed molecular weight in experimental systems . This relatively large size impacts detection methodologies in several ways:

When performing Western blot analysis, researchers should be aware that a band at approximately 100 kDa is expected. This has been confirmed in multiple cell lines including HeLa, U251, KNRK, and NIH3T3 . Understanding the molecular weight is crucial for accurately interpreting results and distinguishing specific binding from non-specific interactions or cross-reactivity.

What are the optimal dilutions and conditions for using PSMD2 antibody in Western blotting?

For optimal results in Western blotting applications with PSMD2 antibody, researchers should follow these methodological guidelines:

The recommended dilution ranges for PSMD2 antibody in Western blotting applications vary by product but typically fall between 1:1000 to 1:4000 . For specific antibodies like 14748-1-AP, this range has been validated across multiple cell lines including SKOV-3, HeLa, K-562, PC-3, A431, and HL-60 cells, as well as in tissue samples from human heart, mouse skeletal muscle, mouse heart, and rat heart .

When optimizing Western blot protocols:

• Sample preparation is critical: Use proper lysis buffers containing protease inhibitors to prevent degradation of PSMD2.

• Loading concentration: 35 μg of total protein per lane has been shown to be effective for detecting PSMD2 in cell lysates .

• Detection method: Chemiluminescence detection provides sufficient sensitivity for visualizing PSMD2 bands .

• Blocking conditions: 5% non-fat milk in TBST is typically sufficient, but BSA may be preferred if phosphorylation states are being studied.

It is important to note that the antibody should be titrated in each testing system to obtain optimal results, as performance can vary depending on the specific sample type and experimental conditions .

How should researchers prepare samples for immunohistochemistry using PSMD2 antibody?

For successful immunohistochemistry (IHC) using PSMD2 antibody, sample preparation is crucial and involves several key steps:

• Fixation: Standard formalin fixation is compatible with most PSMD2 antibodies, but overfixation should be avoided as it can mask epitopes.

• Antigen retrieval: This is a critical step for PSMD2 detection:

  • Primary recommendation: TE buffer at pH 9.0

  • Alternative method: Citrate buffer at pH 6.0

• Dilution optimization: For IHC applications, a dilution range of 1:50-1:500 is typically recommended . The exact dilution should be determined empirically for each tissue type.

• Controls: Positive controls such as human breast cancer tissue have been validated for PSMD2 detection . Including a negative control (omitting primary antibody) is essential to identify any non-specific staining.

• Blocking: Thorough blocking of endogenous peroxidase activity and non-specific binding sites is essential before antibody application.

• Incubation conditions: Overnight incubation at 4°C often yields better results than shorter incubations at room temperature.

The detection of PSMD2 in IHC typically reveals both cytoplasmic and some nuclear staining patterns, consistent with its function in the ubiquitin-proteasome system which operates in both cellular compartments .

What approaches can be used to validate PSMD2 antibody specificity in experimental systems?

Validating antibody specificity is essential for ensuring reliable research results. For PSMD2 antibody, several complementary approaches can be employed:

• Knockout/Knockdown validation: Several PSMD2 antibodies have been validated in KD/KO systems as referenced in the literature . This is considered the gold standard for antibody validation as it demonstrates specificity against the intended target.

• Multi-antibody approach: Using antibodies targeting different epitopes of PSMD2 and comparing staining patterns. If both antibodies show similar results, specificity is more likely.

• Western blot analysis: A single, clear band at the expected molecular weight of 100 kDa should be observed across different cell types and tissues . Multiple or unexpected bands may indicate cross-reactivity.

• Peptide competition assay: Pre-incubating the antibody with the immunogen peptide should abolish specific staining.

• Cross-species reactivity analysis: Examining whether the reactivity pattern matches the expected evolutionary conservation of PSMD2. The antibody should detect PSMD2 in species with high sequence homology (human, mouse, rat) as confirmed for most PSMD2 antibodies .

• Immunoprecipitation followed by mass spectrometry: This can definitively identify the proteins being recognized by the antibody.

Implementing multiple validation approaches strengthens confidence in antibody specificity and experimental results.

How can PSMD2 antibody be effectively utilized in immunofluorescence and confocal microscopy studies?

PSMD2 antibodies can be powerful tools for immunofluorescence (IF) and confocal microscopy studies, providing insights into the subcellular localization and co-localization with other proteins. Based on validated protocols, researchers should consider the following methodological approaches:

• Cell preparation: Fix cells with paraformaldehyde (typically 4%) and permeabilize with a gentle detergent such as 0.15% Triton X-100 to maintain cellular architecture while allowing antibody access .

• Antibody concentration: A working concentration of approximately 10 μg/mL has been shown to be effective for IF applications with PSMD2 antibodies .

• Secondary antibody selection: Fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 488) at 2 μg/mL provide good signal-to-noise ratio .

• Nuclear counterstaining: DAPI is commonly used to visualize nuclei and helps determine whether PSMD2 shows nuclear localization in addition to cytoplasmic distribution .

• Controls: Include negative controls using non-immune IgG from the same species as the primary antibody at equivalent concentrations to identify any non-specific binding .

Immunofluorescence studies with PSMD2 antibody typically reveal cytoplasmic staining with some nuclear localization, consistent with the protein's role in the ubiquitin-proteasome system that functions throughout the cell . This staining pattern has been validated in various cell types including HeLa cells . Advanced co-localization studies can be performed to investigate PSMD2's interaction with other proteasome components or potential substrate proteins.

What methodologies are recommended for studying PSMD2 interactions using co-immunoprecipitation?

Co-immunoprecipitation (Co-IP) is a valuable technique for investigating protein-protein interactions involving PSMD2, particularly its associations within the 26S proteasome complex and with tumor necrosis factor receptors. Based on validated protocols, researchers should consider these methodological approaches:

• Antibody selection: PSMD2 antibodies validated for IP applications, such as 14748-1-AP, have demonstrated successful results in cell lines like K-562 .

• Antibody amount: For effective immunoprecipitation, 0.5-4.0 μg of antibody is recommended for every 1.0-3.0 mg of total protein lysate . Optimization may be necessary depending on the expression level of PSMD2 in your specific system.

• Lysis conditions: Use mild, non-denaturing lysis buffers to preserve protein-protein interactions. RIPA buffer with reduced detergent concentration or NP-40 buffer (1%) is often suitable.

• Pre-clearing: Pre-clear lysates with control IgG and protein A/G beads to reduce non-specific binding.

• Incubation conditions: Overnight incubation at 4°C with gentle rotation typically yields better results than shorter incubations.

• Washing stringency: Multiple gentle washes are crucial to reduce background while preserving specific interactions.

• Detection: Western blotting for potential interaction partners is the standard detection method after Co-IP.

For studying PSMD2's role in the proteasome complex, researchers can investigate its interactions with other proteasome subunits. Additionally, Co-IP can be used to study PSMD2's binding to the intracellular domain of tumor necrosis factor type 1 receptor, as this interaction has been specifically noted in the literature and occurs outside the death domain of TNFR1 .

How can flow cytometry be optimized for detecting PSMD2 in different cell populations?

Flow cytometry allows researchers to quantitatively analyze PSMD2 expression at the single-cell level across heterogeneous populations. Based on validated protocols, the following methodological considerations should be addressed:

• Cell fixation and permeabilization: PSMD2 is an intracellular protein, requiring effective permeabilization. Paraformaldehyde fixation followed by permeabilization with 0.5% Triton X-100 has been validated for PSMD2 detection .

• Antibody concentration: For intracellular flow cytometry (Flow Cyt Intra), a working concentration of approximately 10 μg/mL of PSMD2 antibody has proven effective .

• Secondary antibody selection: Fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 488) at a concentration of 1 μg/mL provide good signal detection .

• Controls: Include isotype controls (e.g., unimmunized goat or rabbit IgG) at the same concentration as the primary antibody to establish gating strategies and identify non-specific binding .

• Compensation: When using multiple fluorophores, proper compensation is essential to account for spectral overlap.

• Analysis considerations:

  • For cells where PSMD2 expression varies with cell cycle, correlation with DNA content staining can provide additional insights

  • For tissues with heterogeneous cell populations, combining PSMD2 staining with cell-type-specific markers allows population-specific analysis

Flow cytometry has been successfully applied to detect PSMD2 in cell lines such as HeLa cells , and this approach can be adapted to study PSMD2 expression in primary cells, patient samples, or sorted cell populations to understand its role in normal physiology and disease states.

What are common challenges in Western blotting with PSMD2 antibody and how can they be resolved?

Researchers frequently encounter several challenges when performing Western blots for PSMD2 detection. These issues and their solutions include:

• Weak or absent signal:

  • Cause: Insufficient protein loading, inadequate antibody concentration, or poor transfer efficiency of high molecular weight proteins

  • Solution: Increase protein loading (35 μg per lane has been validated) ; optimize antibody concentration within the recommended 1:1000-1:4000 range ; extend transfer time for the 100 kDa PSMD2 protein; use lower percentage gels (8-10%)

• Multiple bands or non-specific binding:

  • Cause: Cross-reactivity, protein degradation, or post-translational modifications

  • Solution: Increase blocking stringency; optimize antibody dilution; include protease inhibitors during sample preparation; validate with positive controls such as SKOV-3, HeLa, or K-562 cell lysates

• Inconsistent band intensity across samples:

  • Cause: Uneven loading, transfer issues, or variable PSMD2 expression

  • Solution: Normalize to housekeeping proteins; ensure even transfer using stain-free technology or Ponceau staining; load equal amounts of protein

• Background issues:

  • Cause: Insufficient blocking, inappropriate antibody concentration, or poor washing

  • Solution: Optimize blocking conditions; increase washing duration and volume; adjust secondary antibody concentration

• Band at unexpected molecular weight:

  • Cause: Post-translational modifications, splice variants, or non-specific binding

  • Solution: Confirm with additional PSMD2 antibodies targeting different epitopes; perform validation studies using PSMD2 knockdown or knockout samples

When interpreting Western blot results, researchers should expect to observe a band at approximately 100 kDa, which is both the calculated and observed molecular weight for PSMD2 . This has been consistently observed across various cell lines and tissue samples .

How should researchers interpret variable staining patterns in immunohistochemistry using PSMD2 antibody?

Interpreting PSMD2 staining patterns in immunohistochemistry requires understanding the biological context and potential sources of variability. Researchers should consider these key points:

• Expected staining pattern: PSMD2 typically shows cytoplasmic and some nuclear staining, consistent with its role in the ubiquitin-proteasome system that functions in both compartments . Validated positive controls like human breast cancer tissue can help establish expected patterns .

• Tissue-specific variations:

  • PSMD2 is widely expressed across tissues including skeletal muscle, liver, heart, brain, kidney, pancreas, lung, and placenta

  • Expression levels naturally vary between tissues, which should not be misinterpreted as technical artifacts

  • Complete absence of staining in tissues known to express PSMD2 likely indicates technical issues

• Interpreting heterogeneous staining:

  • Cell-type specific expression within a tissue is biologically meaningful

  • Correlation with proliferation markers may be relevant as PSMD2 is involved in cell cycle regulation

  • Gradients of expression may reflect physiological or pathological states

• Technical sources of variability:

  • Antigen retrieval efficiency: Compare TE buffer pH 9.0 (recommended) versus citrate buffer pH 6.0 (alternative)

  • Antibody dilution: Optimize within the recommended range (1:50-1:500)

  • Fixation time and conditions can significantly impact epitope availability

  • Detection system sensitivity affects signal intensity

• Validation approaches for uncertain results:

  • Compare with mRNA expression data from public databases

  • Perform parallel staining with another validated PSMD2 antibody targeting a different epitope

  • Include positive and negative control tissues in the same experimental run

Understanding both technical and biological sources of variability allows researchers to distinguish meaningful differences in PSMD2 expression from artifacts.

What are the critical considerations for quantifying PSMD2 expression in experimental systems?

Accurate quantification of PSMD2 expression is essential for comparative studies across experimental conditions. Researchers should consider these methodological approaches:

• Western blot quantification:

  • Always include loading controls appropriate for your experimental system

  • Use digital image analysis software that accounts for signal saturation

  • Establish a standard curve with recombinant protein or cell lysates with known PSMD2 content

  • Report relative expression normalized to controls rather than absolute values

• Immunohistochemistry quantification:

  • Use digital pathology approaches with validated algorithms for cellular compartment separation

  • Consider both staining intensity and percentage of positive cells (H-score or Allred score)

  • Blind analysis by multiple observers reduces bias

  • Compare with orthogonal methods like Western blotting from the same samples

• Flow cytometry quantification:

  • Use median fluorescence intensity rather than mean when distributions are non-normal

  • Include calibration beads to allow comparison between experimental runs

  • Apply consistent gating strategies based on isotype controls

  • Consider using quantitative flow cytometry with beads of known antibody binding capacity

• qPCR correlation:

  • While measuring PSMD2 mRNA levels can provide complementary data, protein levels may not directly correlate due to post-transcriptional regulation

  • Use validated reference genes appropriate for your experimental system

  • Perform protein-mRNA correlation studies to understand the relationship in your specific model

• Normalization considerations:

  • For tissue samples, normalize to total protein rather than single housekeeping proteins

  • For cell cycle-dependent studies, consider synchronizing cells or correlating with cell cycle markers

  • For stress response studies, measure multiple time points as proteasome components may change dynamically

These quantification approaches should be tailored to the specific research question and experimental system under investigation.

How is PSMD2 antibody being utilized in cancer research studies?

PSMD2 antibody has become an important tool in cancer research due to the critical role of the ubiquitin-proteasome system in tumor biology. Key research applications include:

• Expression profiling: PSMD2 antibody has been used to examine expression patterns in various cancer types including breast cancer, where it serves as a validated positive control for immunohistochemistry . Researchers investigate whether PSMD2 expression correlates with cancer progression, metastasis, or patient outcomes.

• Mechanistic studies: As a component of the 26S proteasome, PSMD2 participates in degrading proteins involved in cell cycle regulation, apoptosis, and DNA damage repair . Cancer researchers use PSMD2 antibody to study how alterations in proteasomal activity contribute to cancer cell survival and resistance to therapy.

• Proteasome inhibitor research: Proteasome inhibitors like bortezomib are used clinically for treating certain cancers. PSMD2 antibody helps researchers investigate the molecular mechanisms behind sensitivity or resistance to these drugs by examining changes in proteasome complex composition or post-translational modifications.

• Interaction studies: Co-immunoprecipitation with PSMD2 antibody allows researchers to identify cancer-specific interaction partners that may reveal novel therapeutic targets .

• Localization studies: Immunofluorescence using PSMD2 antibody reveals subcellular distribution patterns in cancer cells versus normal cells, potentially uncovering cancer-specific alterations in proteasome localization or function .

Researchers studying PSMD2 in cancer contexts should select antibodies validated in relevant cancer models and consider combining multiple detection methods (IHC, WB, IF) to strengthen their findings.

What methodological approaches can be used to study PSMD2 in the context of neurodegenerative diseases?

The ubiquitin-proteasome system plays a crucial role in neurodegenerative diseases characterized by protein aggregation. Researchers investigating PSMD2 in this context should consider these methodological approaches:

• Brain tissue analysis:

  • Immunohistochemistry with PSMD2 antibody can reveal alterations in expression or localization in affected brain regions

  • Double-labeling with markers of neuronal subtypes or aggregated proteins (tau, α-synuclein, etc.) provides context-specific information

  • Recommended dilution for IHC (1:50-1:500) should be optimized for brain tissue

• Co-localization studies:

  • Immunofluorescence combining PSMD2 antibody with markers of protein aggregates can reveal whether the proteasome is recruited to these structures

  • Confocal microscopy with z-stack analysis provides three-dimensional information about spatial relationships

• Biochemical fractionation:

  • Western blotting with PSMD2 antibody (1:1000-1:4000 dilution) can detect shifts between soluble and insoluble protein fractions

  • Sequential extraction protocols can separate proteins based on solubility, revealing whether PSMD2 becomes sequestered in aggregates

• Animal models:

  • Transgenic models of neurodegeneration can be analyzed for changes in PSMD2 expression, localization, or association with other proteins

  • Age-dependent changes may be particularly relevant to neurodegenerative processes

• Cell culture models:

  • Primary neurons treated with aggregation-prone proteins or subjected to stressors relevant to neurodegeneration

  • Co-immunoprecipitation to identify altered interactions with PSMD2 under pathological conditions

When interpreting results, researchers should consider that proteasome dysfunction may be both a cause and consequence of protein aggregation in neurodegenerative diseases, requiring careful experimental design to distinguish these possibilities.

How can PSMD2 antibody contribute to understanding post-translational modifications of proteasome components?

Post-translational modifications (PTMs) of proteasome components, including PSMD2, play crucial roles in regulating proteasome assembly, localization, and activity. PSMD2 antibody can be instrumental in studying these modifications through several methodological approaches:

• PTM-specific detection strategies:

  • Using general PSMD2 antibody for immunoprecipitation followed by PTM-specific antibodies for detection

  • Combining PSMD2 immunoprecipitation with mass spectrometry to comprehensively identify PTMs

  • 2D gel electrophoresis with PSMD2 antibody detection to separate modified forms based on charge and size

• Functional impact assessment:

  • Comparing native versus modified PSMD2 in co-immunoprecipitation assays to identify altered protein interactions

  • Immunofluorescence to determine whether PTMs affect subcellular localization

  • Activity assays after immunoprecipitation to link specific modifications with proteasome function

• PTM dynamics investigation:

  • Western blotting with PSMD2 antibody across time courses after cellular stimulation

  • Phosphatase or deubiquitinase treatments prior to Western blotting to confirm specific modifications

  • Comparison across tissues or cell types to identify context-specific regulation

• Technical considerations for PTM studies:

  • Extraction conditions must preserve the modifications of interest (phosphatase inhibitors for phosphorylation, deubiquitinase inhibitors for ubiquitination)

  • Antibody selection: ensure the PSMD2 antibody's epitope does not overlap with or contain the modification site of interest

  • Consider whether modifications affect antibody recognition, potentially leading to false negative results

These approaches enable researchers to understand how PTMs regulate PSMD2 and the entire proteasome complex in health and disease. When properly employed, PSMD2 antibody becomes a powerful tool for elucidating the complex regulatory networks controlling protein homeostasis through the ubiquitin-proteasome system.