PSMD11 PAT2C7AT Antibody

What is PSMD11 and what is its biological significance?

PSMD11 (26S proteasome non-ATPase regulatory subunit 11) is a critical component of the 26S proteasome, a multiprotein complex involved in the ATP-dependent degradation of ubiquitinated proteins. This complex maintains protein homeostasis by removing misfolded or damaged proteins that could impair cellular functions and by eliminating proteins whose functions are no longer required. PSMD11 specifically functions in proteasome assembly and plays a key role in enhancing proteasome activity in embryonic stem cells (ESCs) .

PSMD11 is also known by several other names including 26S proteasome regulatory subunit RPN6, 26S proteasome regulatory subunit S9, and 26S proteasome regulatory subunit p44.5 . Structurally, PSMD11 consists of 422 amino acids and contains a PCI domain essential for its function in ATP-dependent degradation pathways . The gene encoding PSMD11 is located on human chromosome 17, a region that constitutes over 2.5% of the human genome and encodes more than 1,200 genes, highlighting PSMD11's importance in cellular control and proteostasis .

What are the key characteristics of the PAT2C7AT antibody?

The PAT2C7AT antibody is a mouse monoclonal antibody specifically designed to target PSMD11. Its key characteristics include:

The antibody is derived from hybridization of mouse F0 myeloma cells with spleen cells from BALB/c mice immunized with the full-length recombinant human PSMD11 protein .

How should the PAT2C7AT antibody be stored to maintain optimal activity?

Proper storage of the PAT2C7AT antibody is crucial for maintaining its specificity and activity. The recommended storage conditions are:

• Short-term storage (up to 1 month): Store at 2-8°C (standard refrigeration)

• Long-term storage: Store at -20°C

• Important note: Avoid repeated freeze-thaw cycles as they can significantly degrade antibody quality and performance

When shipping, the antibody should be transported with ice packs to maintain stability . Before use, thaw the antibody completely and mix gently to ensure homogeneity. Aliquoting the antibody upon first thaw is recommended if multiple uses are planned to avoid repeated freeze-thaw cycles.

What are the optimal working dilutions for different applications?

The optimal working dilutions for the PAT2C7AT antibody vary depending on the specific application:

These recommendations serve as starting points. Optimal dilutions should be determined empirically for each specific application and experimental system. Performing antibody titration experiments is strongly recommended to identify the concentration that provides the best signal-to-noise ratio for your particular samples and detection methods.

How can I validate the specificity of the PAT2C7AT antibody in my experimental system?

Validating antibody specificity is critical for obtaining reliable results. For the PAT2C7AT antibody, consider these validation approaches:

• Western blot validation:

  • Confirm detection of a single band at approximately 47 kDa (the expected molecular weight of PSMD11)

  • Test in multiple cell lines with known PSMD11 expression levels

  • Compare with patterns obtained using other validated PSMD11 antibodies

• Genetic validation:

  • Use PSMD11 knockdown/knockout samples to confirm signal reduction/elimination

  • Perform PSMD11 overexpression to demonstrate increased signal intensity

  • If possible, use cells from different species to confirm cross-reactivity claims

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • Observe elimination of specific signal in subsequent applications

• Orthogonal methods:

  • Confirm PSMD11 detection using mass spectrometry following immunoprecipitation

  • Perform RT-PCR to correlate protein detection with mRNA expression levels

• Application-specific controls:

  • For IF, include secondary-only controls to assess background

  • For flow cytometry, use isotype controls to determine non-specific binding

  • For IP, use IgG2b isotype control to identify non-specific pull-down

Implementing these validation strategies will increase confidence in the specificity of the PAT2C7AT antibody and the reliability of subsequent experimental results.

How does the PAT2C7AT antibody compare to other PSMD11 antibodies?

Understanding the differences between available PSMD11 antibodies can help researchers select the most appropriate reagent for their specific applications:

The choice between these antibodies should consider:

• Application compatibility: Select an antibody validated for your specific application

• Species compatibility: Ensure reactivity with your experimental model

• Isotype considerations: Different isotypes may perform better in specific applications (e.g., IgG2b may offer advantages for certain detection systems)

• Validation status: Consider the extent of validation data available for each antibody

• Experimental workflow: Choose antibodies compatible with your detection systems and other reagents

For multiplexing experiments, combining antibodies from different host species (e.g., PAT2C7AT mouse antibody with rabbit antibodies against other targets) can facilitate simultaneous detection of multiple proteins .

How can I use the PAT2C7AT antibody to study proteasome assembly?

PSMD11 is required for proteasome assembly, making the PAT2C7AT antibody valuable for investigating this process. Consider these methodological approaches:

• Co-immunoprecipitation (Co-IP) studies:

  • Use PAT2C7AT to pull down PSMD11 and analyze co-precipitating proteins

  • Identify interaction partners during different assembly stages

  • Compare assembly complexes under various cellular conditions (stress, differentiation, etc.)

• Density gradient centrifugation with immunoblotting:

  • Separate proteasome assembly intermediates by sucrose gradient ultracentrifugation

  • Use PAT2C7AT in Western blots to track PSMD11 across gradient fractions

  • Monitor shifts in PSMD11 distribution in response to assembly perturbations

• Native gel electrophoresis:

  • Separate intact proteasome complexes on native PAGE gels

  • Detect PSMD11-containing complexes using PAT2C7AT

  • Perform in-gel proteasome activity assays to correlate assembly with function

• Immunofluorescence microscopy:

  • Visualize PSMD11 localization during different stages of proteasome assembly

  • Perform co-localization studies with other proteasome subunits

  • Track dynamic changes in PSMD11 distribution during cellular stress responses

• Pulse-chase analysis:

  • Track newly synthesized PSMD11 incorporation into maturing proteasomes

  • Immunoprecipitate with PAT2C7AT at different time points after metabolic labeling

  • Analyze the kinetics of proteasome assembly in different cell types or conditions

These approaches can provide comprehensive insights into PSMD11's role in proteasome assembly and how this process might be regulated under different physiological or pathological conditions .

What is the significance of PSMD11 in stem cell research and how can PAT2C7AT antibody contribute?

PSMD11 plays a key role in enhancing proteasome activity in embryonic stem cells (ESCs), making it an important factor in stem cell biology. The PAT2C7AT antibody can be utilized to investigate this connection through:

• Comparative expression analysis:

  • Use Western blotting with PAT2C7AT to quantify PSMD11 levels across:

    • Pluripotent vs. differentiated cells

    • Different stages of cellular reprogramming

    • Various differentiation lineages

  • Correlate PSMD11 levels with proteasome activity measurements

• Regulatory mechanism studies:

  • Investigate FOXO4-mediated regulation of PSMD11 expression in human ESCs

  • Combine chromatin immunoprecipitation (ChIP) for transcription factors with PAT2C7AT detection

  • Analyze how pluripotency factors influence PSMD11 expression

• Proteasome assembly in stem cells:

  • Compare proteasome assembly efficiency between stem cells and differentiated cells

  • Use PAT2C7AT with native gel electrophoresis to assess PSMD11 incorporation

  • Analyze whether PSMD11 is rate-limiting for proteasome assembly in different cell types

• Functional studies:

  • Manipulate PSMD11 levels in stem cells and assess effects on:

    • Pluripotency maintenance

    • Differentiation capacity

    • Protein quality control

    • Response to proteotoxic stress

  • Use PAT2C7AT to confirm knockdown or overexpression efficiency

Understanding PSMD11's role in stem cells could provide insights into how protein homeostasis mechanisms contribute to pluripotency maintenance and differentiation processes, potentially informing regenerative medicine approaches .

What approaches can be used to study PSMD11 post-translational modifications?

Post-translational modifications (PTMs) can regulate PSMD11 function and proteasome assembly. Here are methodological approaches using PAT2C7AT antibody to study PSMD11 PTMs:

• Phosphorylation analysis:

  • Immunoprecipitate PSMD11 using PAT2C7AT

  • Analyze by phospho-specific Western blotting or mass spectrometry

  • Compare phosphorylation patterns after treatment with kinase inhibitors

  • Perform λ-phosphatase treatment to confirm phosphorylation-dependent mobility shifts

• Ubiquitination studies:

  • Perform denaturing immunoprecipitation with PAT2C7AT

  • Probe for ubiquitin to detect ubiquitinated PSMD11

  • Use proteasome inhibitors to enhance detection of ubiquitinated forms

  • Compare ubiquitination patterns under different cellular stresses

• Two-dimensional gel electrophoresis:

  • Separate proteins by isoelectric point and molecular weight

  • Detect PSMD11 isoforms using PAT2C7AT

  • Identify PTM-dependent shifts in PSMD11 migration patterns

  • Compare PTM profiles across different cell types or conditions

• PSMD11 PTM-function correlation:

  • Generate PTM-mimetic or PTM-deficient PSMD11 mutants

  • Compare their abilities to support proteasome assembly

  • Use PAT2C7AT to assess incorporation of mutants into proteasome complexes

  • Correlate identified PTMs with proteasome assembly efficiency and activity

• Mass spectrometry-based PTM mapping:

  • Immunoprecipitate PSMD11 using PAT2C7AT

  • Perform high-resolution mass spectrometry analysis

  • Create a comprehensive map of PSMD11 PTMs

  • Monitor dynamic changes in PTM patterns during cellular processes

These approaches can reveal how PSMD11 function is regulated at the post-translational level and how these modifications might impact proteasome assembly and activity in different cellular contexts .

Why might I observe non-specific bands in Western blots, and how can I address this issue?

Non-specific bands in Western blots using PAT2C7AT antibody can complicate data interpretation. Here are potential causes and solutions:

Potential Causes:

• Cross-reactivity with related proteins:

  • PSMD11 belongs to a family of proteasome subunits with sequence homology

  • The antibody might recognize shared epitopes in related proteins

• Sample preparation issues:

  • Protein degradation during sample preparation

  • Incomplete denaturation of protein complexes

  • Sample overloading leading to non-specific binding

• Technical factors:

  • Insufficient blocking

  • Overly sensitive detection systems

  • Extended exposure times amplifying background signals

Methodological Solutions:

• Optimize blocking conditions:

  • Test different blocking agents (5% BSA, 5% milk, commercial blockers)

  • Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Add 0.05-0.1% Tween-20 to reduce non-specific hydrophobic interactions

• Adjust antibody dilution and incubation:

  • Try more dilute antibody solution (e.g., 1:2000 instead of 1:1000)

  • Incubate primary antibody at 4°C overnight rather than at room temperature

  • Prepare antibody in fresh blocking solution

• Improve sample preparation:

  • Add protease inhibitor cocktail to prevent degradation

  • Ensure complete denaturation (boil samples for 5-10 minutes in SDS buffer)

  • Optimize protein loading (10-30 μg for cell lysates)

  • Centrifuge samples after boiling to remove insoluble material

• Enhance washing:

  • Increase number of washes (5-6 times for 5-10 minutes each)

  • Use larger volumes of wash buffer

  • Add additional Tween-20 (0.1-0.2%) to wash buffers

• Validate with controls:

  • Run PSMD11 knockdown samples to identify specific bands

  • Include molecular weight markers to accurately identify the expected 47 kDa band

  • Compare results with another validated PSMD11 antibody

These optimizations should help distinguish the specific PSMD11 signal (~47 kDa) from non-specific bands, improving data quality and interpretation .

What are the critical parameters for successful immunoprecipitation using PAT2C7AT antibody?

Successful immunoprecipitation (IP) with PAT2C7AT antibody requires careful optimization of several critical parameters:

• Cell lysis optimization:

  • Use buffers compatible with maintaining protein-protein interactions

  • Common IP buffers include RIPA (more stringent) or NP-40/Triton X-100 (milder)

  • Include protease inhibitors and phosphatase inhibitors if studying phosphorylation

  • Lysis buffer composition example:

    • 50 mM Tris-HCl (pH 7.4)

    • 150 mM NaCl

    • 1% NP-40 or 0.5% Triton X-100

    • 1 mM EDTA

    • Protease inhibitor cocktail

• Antibody amount optimization:

  • Typically use 1-5 μg antibody per 500-1000 μg of protein lysate

  • Perform titration experiments to determine optimal antibody concentration

  • Insufficient antibody leads to poor target recovery

  • Excess antibody can increase non-specific binding

• Pre-clearing step:

  • Incubate lysate with protein A/G beads before adding antibody

  • Removes proteins that bind non-specifically to beads

  • Typically 1 hour at 4°C with gentle rotation

• Immunoprecipitation procedure:

  • Add optimized amount of PAT2C7AT antibody to pre-cleared lysate

  • Incubate 2-4 hours or overnight at 4°C with gentle rotation

  • Add pre-washed protein A/G beads (40-50 μl of 50% slurry)

  • Incubate 1-2 hours at 4°C with gentle rotation

• Washing optimization:

  • Perform 4-5 washes with lysis buffer

  • Consider increasing salt concentration in middle washes (up to 300 mM NaCl)

  • Gentle pelleting of beads (1000-2000 × g for 1 minute)

  • Careful removal of supernatant without disturbing bead pellet

• Elution considerations:

  • For Western blot analysis: add 2× Laemmli buffer and boil for 5 minutes

  • For native conditions: consider specific peptide elution or mild acid elution

• Critical controls:

  • Input sample (5-10% of lysate used for IP)

  • IgG2b isotype control processed identically to experimental samples

  • IP in PSMD11 knockdown cells if available

Following these guidelines should allow for specific immunoprecipitation of PSMD11 using the PAT2C7AT antibody, facilitating the study of PSMD11 interactions and modifications .

How can I optimize immunofluorescence staining using the PAT2C7AT antibody?

Immunofluorescence (IF) with the PAT2C7AT antibody requires careful optimization to achieve specific PSMD11 detection with minimal background:

• Fixation optimization:

  • Compare different fixation methods:

    • 4% paraformaldehyde (10-15 minutes at room temperature)

    • Ice-cold methanol (10 minutes at -20°C)

    • Combination approaches (PFA followed by methanol)

  • PSMD11 detection may be sensitive to fixation method due to epitope accessibility

• Permeabilization considerations:

  • Test different permeabilization agents:

    • 0.1-0.25% Triton X-100 in PBS (10 minutes)

    • 0.5% Saponin in PBS (15 minutes)

    • 0.05% Tween-20 in PBS (15 minutes)

  • Balance permeabilization efficiency with preservation of cellular structures

• Blocking optimization:

  • Use 5-10% normal serum (from secondary antibody host species)

  • Add 1-3% BSA to reduce non-specific binding

  • Consider adding 0.1-0.3% Triton X-100 to blocking solution

  • Block for 30-60 minutes at room temperature or overnight at 4°C

• Antibody dilution optimization:

  • Start with 1:100-1:500 dilution of PAT2C7AT

  • Prepare antibody in blocking buffer with reduced serum (1-2%)

  • Incubate overnight at 4°C in a humidified chamber

  • For co-staining, select compatible antibodies from different host species

• Washing protocol:

  • Perform at least 3 washes with PBS

  • Add 0.05% Tween-20 to wash buffer

  • Wash for 5-10 minutes per wash

  • Use gentle agitation during washing

• Signal detection considerations:

  • Select secondary antibody with appropriate conjugate

  • Use anti-mouse IgG2b-specific secondary if possible

  • Consider tyramide signal amplification for low abundance signals

  • Use appropriate mounting medium with anti-fade properties

• Critical controls:

  • Secondary antibody only (omit primary antibody)

  • IgG2b isotype control at same concentration as PAT2C7AT

  • DAPI or Hoechst nuclear counterstain to assess cell morphology

  • If possible, PSMD11 knockdown cells as negative control

PSMD11 is expected to show both nuclear and cytoplasmic localization, with potential enrichment in areas of active proteasome assembly. The staining pattern should be validated using orthogonal approaches such as subcellular fractionation followed by Western blotting .

What are the key considerations for flow cytometry applications with PAT2C7AT antibody?

Flow cytometry with the PAT2C7AT antibody presents unique challenges since PSMD11 is primarily an intracellular protein. Here are key methodological considerations:

• Cell preparation and fixation:

  • Harvest cells gently to maintain viability (trypsin or EDTA)

  • Wash thoroughly in PBS to remove media components

  • Fix with 2-4% paraformaldehyde for 10-15 minutes at room temperature

  • Alternative: fix with 70-80% ice-cold methanol for 30 minutes at -20°C

• Permeabilization optimization:

  • For PFA-fixed cells: permeabilize with 0.1-0.5% saponin or 0.1-0.3% Triton X-100

  • For methanol-fixed cells: additional permeabilization may not be necessary

  • Saponin requires continuous presence in all buffers to maintain permeabilization

• Blocking and antibody staining:

  • Block with 2-5% BSA or 5-10% normal serum

  • Start with 1:100 dilution of PAT2C7AT antibody

  • Incubate for 30-60 minutes at room temperature or overnight at 4°C

  • Use fluorophore-conjugated anti-mouse IgG2b secondary antibody

  • For multicolor panels, consider directly conjugating PAT2C7AT

• Critical controls:

  • Unstained cells to establish autofluorescence baseline

  • Secondary antibody only to assess background staining

  • IgG2b isotype control at the same concentration as PAT2C7AT

  • If available, PSMD11 knockdown cells as biological negative control

• Acquisition and analysis considerations:

  • Collect sufficient events (minimum 10,000-20,000 per sample)

  • Gate on intact cells using FSC/SSC

  • Exclude doublets using FSC-H vs. FSC-A

  • Use viability dye to exclude dead cells (if not using methanol fixation)

  • For analysis, compare median fluorescence intensity (MFI) rather than percent positive

  • PSMD11 will likely show a shift in the entire population rather than distinct positive/negative populations

• Data interpretation:

  • Consider PSMD11 expression heterogeneity across cell cycle phases

  • Compare MFI across different experimental conditions

  • For meaningful comparisons, maintain consistent instrument settings

  • Perform replicate experiments to ensure reproducibility

With proper optimization, flow cytometry with PAT2C7AT antibody can provide quantitative information about PSMD11 expression levels across different cell populations or treatment conditions .