PSMB10 Antibody

What is PSMB10 and what is its role in the proteasome system?

PSMB10 (Proteasome Subunit Beta Type-10), also known as MECL-1 or LMP10, is a component of the immunoproteasome, which is a specialized form of the 20S proteasome. It is expressed as a proenzyme (29 kDa) that undergoes autocatalytic cleavage to form a mature immunoproteasome core particle subunit (25 kDa) . Unlike constitutive proteasomes, PSMB10 expression is induced by IFN-γ, where it replaces the constitutively expressed PSMB7/Z subunit within the 20S proteasome proteolytic core particle . This replacement modifies the cleavage specificity of the proteasome, facilitating the processing and presentation of MHC class I-restricted peptide antigens on the cell surface . The immunoproteasome plays a crucial role in protecting cells against the accumulation of oxidant-damaged proteins and contributes to immune surveillance .

What are the common applications for PSMB10 antibodies?

PSMB10 antibodies are versatile tools utilized in multiple research applications:

For optimal results, researchers should validate the antibody in their specific experimental system and adjust dilutions accordingly .

How can I confirm the specificity of my PSMB10 antibody?

To validate PSMB10 antibody specificity:

• Knockout validation: Use PSMB10 knockout cell lines (such as PSMB10 KO HeLa cells) alongside wild-type controls to confirm absence of signal in knockout samples .

• Cross-reactivity assessment: Test the antibody against related proteasome subunits to ensure no cross-reactivity with other beta-type subunits.

• Molecular weight verification: Confirm detection at the expected molecular weight (~25-29 kDa) .

• Peptide competition: Pre-incubate the antibody with the immunizing peptide and observe signal reduction.

• Multiple detection methods: Validate specificity across different applications (WB, IHC, IF) to ensure consistent results .

How can PSMB10 antibodies be used to study immunoproteasome assembly and regulation?

PSMB10 antibodies can provide valuable insights into immunoproteasome assembly and regulation through several methodological approaches:

• Co-immunoprecipitation studies: Use PSMB10 antibodies to pull down the entire immunoproteasome complex and analyze associated proteins to understand assembly intermediates and regulatory factors.

• Pulse-chase experiments: Combine PSMB10 antibodies with metabolic labeling to track the synthesis, maturation, and turnover of immunoproteasome subunits.

• IFN-γ induction kinetics: Use time-course experiments with PSMB10 antibodies to monitor the replacement of constitutive proteasome subunits with immunoproteasome subunits following IFN-γ treatment .

• Subcellular localization studies: Employ immunofluorescence with PSMB10 antibodies to track proteasome distribution under different cellular stresses and stimuli .

• Chromatin immunoprecipitation (ChIP): Utilize PSMB10 antibodies in ChIP experiments to investigate the regulatory mechanisms controlling immunoproteasome gene expression.

What are the technical considerations for using PSMB10 antibodies in studying pathological conditions?

When investigating PSMB10 in disease contexts, several technical considerations are essential:

• Tissue-specific expression: PSMB10 expression varies across tissues, with higher levels in immune-related tissues. Adjust antibody dilutions according to the expected expression level in your target tissue .

• Distinguishing proenzyme vs. mature forms: Select antibodies capable of detecting both the 29 kDa proenzyme and 25 kDa mature form to capture the complete biology .

• Antigen retrieval optimization: For FFPE tissues, test both citrate buffer (pH 6.0) and TE buffer (pH 9.0) antigen retrieval methods to determine optimal conditions .

• Controls for pathological samples: Include both normal tissue controls and disease-relevant positive controls when analyzing pathological specimens.

• Multiplex analysis: Consider dual staining with other immunoproteasome subunits (PSMB8, PSMB9) to assess complete immunoproteasome formation rather than just PSMB10 expression .

How can PSMB10 antibodies be utilized to investigate the role of immunoproteasome in SCID-Omenn syndrome?

Recent research has identified de novo mutations in PSMB10 as a cause of SCID-Omenn syndrome . PSMB10 antibodies can be instrumental in investigating this connection through:

• Patient sample analysis: Apply PSMB10 antibodies to analyze expression levels and localization in patient-derived samples compared to healthy controls.

• Mutation-specific antibodies: Consider developing antibodies that specifically recognize common pathogenic variants (c.166G>C [p.Asp56His] and c.601G>A/c.601G>C [p.Gly201Arg]) .

• Functional studies: Use PSMB10 antibodies to assess how mutations affect:

  • Immunoproteasome assembly

  • Interaction with other subunits

  • Proteolytic activity

  • Stability of the complex

• T cell development analysis: Employ PSMB10 antibodies in combination with T cell markers to investigate how mutations impact T cell maturation and selection .

• Revertant mosaicism detection: Use highly sensitive PSMB10 antibodies to identify cells with genetic reversion that may contribute to partial immune reconstitution .

Western Blotting:

• Lysis buffer optimization: Use RIPA buffer supplemented with protease inhibitors for most applications. For studying proteasome complexes, consider milder NP-40 based buffers to preserve interactions .

• Sample handling: Avoid repeated freeze-thaw cycles as they can disrupt proteasome complexes.

• Denaturation conditions: Heat samples at 95°C for 5 minutes in standard Laemmli buffer with DTT or β-mercaptoethanol .

• Gel percentage: Use 12-15% SDS-PAGE gels for optimal resolution of the 25-29 kDa PSMB10 protein .

Immunohistochemistry:

• Fixation: 10% neutral buffered formalin for 24-48 hours is recommended for most tissues.

• Antigen retrieval: Heat-induced epitope retrieval with TE buffer (pH 9.0) provides optimal results for most PSMB10 antibodies, though citrate buffer (pH 6.0) may be tested as an alternative .

• Blocking: 5-10% normal serum from the same species as the secondary antibody for 1 hour at room temperature.

• Primary antibody incubation: Overnight at 4°C at optimized dilution (typically 1:50-1:600) .

How should experimental controls be designed for PSMB10 antibody validation?

A comprehensive validation strategy should include:

• Positive controls: Select tissues or cell lines with known PSMB10 expression:

  • Lymphoid tissues (spleen, thymus)

  • Immune cell lines (Raji, Daudi, THP-1)

• Negative controls:

  • Primary antibody omission

  • Isotype controls

  • PSMB10 knockout cell lines

• Expression modulation controls:

  • IFN-γ treated vs. untreated cells (should increase PSMB10 expression)

  • Proteasome inhibitor treatment (may affect processing)

• Specificity controls:

  • Peptide competition assays

  • Multiple antibodies targeting different epitopes

  • Correlation with mRNA expression data

• Technical controls:

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

  • Tissue-specific positive controls for IHC

What are the critical parameters for optimizing PSMB10 antibody signal in immunofluorescence experiments?

For optimal immunofluorescence results with PSMB10 antibodies:

• Fixation optimization:

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

  • For certain applications, test methanol fixation (-20°C for 10 minutes)

  • Avoid over-fixation which can mask epitopes

• Permeabilization:

  • 0.1-0.2% Triton X-100 for 10 minutes is suitable for most applications

  • For membrane-associated pools of PSMB10, milder permeabilization with 0.1% saponin may preserve localization

• Antibody dilution and incubation:

  • Recommended dilutions range from 1:400-1:1600

  • Incubate primary antibody at 4°C overnight or 1-2 hours at room temperature

  • Include 1% BSA in antibody diluent to reduce background

• Signal amplification:

  • For weak signals, consider tyramide signal amplification systems

  • Ensure amplification doesn't increase background

• Co-localization studies:

  • Combine PSMB10 antibodies with markers for subcellular compartments

  • Use sequential staining if antibodies are from the same species

Issue: High background in Western blot

Solutions:

• Increase blocking time/concentration (5% non-fat milk or BSA)

• Increase washing steps (5x 5-minute washes with TBST)

• Dilute primary antibody further

• Use a different secondary antibody

• Replace PVDF membrane with nitrocellulose

Issue: Multiple bands in Western blot

Solutions:

• Verify if bands represent proform (29 kDa) and mature form (25 kDa)

• Increase gel percentage for better resolution

• Use fresh protein sample with complete protease inhibitors

• Validate with PSMB10 knockout controls to identify specific bands

Issue: Weak or no signal in IHC

Solutions:

• Optimize antigen retrieval (test both pH 6.0 and pH 9.0 buffers)

• Increase antibody concentration

• Extend primary antibody incubation time

• Use signal amplification methods

• Verify tissue expression levels of PSMB10

How can researchers evaluate the reproducibility of experimental results using PSMB10 antibodies?

To ensure reproducible results:

• Documentation: Maintain detailed records of:

  • Antibody lot number

  • Detailed protocols including exact buffer compositions

  • Image acquisition settings

  • Sample preparation methods

• Quantification methods:

  • Use digital image analysis software for objective quantification

  • Apply consistent thresholding criteria

  • Include calibration standards when possible

• Statistical validation:

  • Perform experiments in biological triplicates minimum

  • Use appropriate statistical tests for small sample sizes

  • Report effect sizes along with p-values

• Cross-validation:

  • Validate key findings with a second PSMB10 antibody targeting a different epitope

  • Confirm with orthogonal methods (mRNA analysis, mass spectrometry)

  • Consider validation in an independent laboratory

• Standardization:

  • Use recombinant PSMB10 as a positive control

  • Include the same control samples across experimental batches

  • Normalize to housekeeping proteins consistently

How are PSMB10 antibodies being used to study the connection between immunoproteasome dysfunction and immune disorders?

PSMB10 antibodies are instrumental in investigating the emerging roles of immunoproteasome in immune disorders:

• Autoimmune disease research: PSMB10 antibodies are being used to study altered immunoproteasome expression and function in conditions like rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis.

• Primary immunodeficiency investigation: The recent discovery of PSMB10 mutations in SCID-Omenn syndrome has opened new avenues for research into immunoproteasome dysfunction in primary immunodeficiencies .

• Transplantation biology: PSMB10 antibodies are valuable for studying the role of immunoproteasome in graft-versus-host disease and transplant rejection.

• Cancer immunotherapy: Researchers are using PSMB10 antibodies to investigate immunoproteasome function in tumor cells and its impact on antigen presentation and immunotherapy response.

• Inflammatory conditions: PSMB10 antibodies help study the contribution of immunoproteasome dysregulation to inflammatory conditions and potential therapeutic interventions targeting this pathway.

What are emerging applications of PSMB10 antibodies in combination with advanced imaging and proteomics techniques?

Recent technological advances have expanded PSMB10 antibody applications:

• Super-resolution microscopy: PSMB10 antibodies compatible with techniques like STORM or PALM allow visualization of immunoproteasome distribution with nanometer precision.

• Proximity labeling: Combining PSMB10 antibodies with BioID or APEX2 proximity labeling systems enables mapping of the immunoproteasome interactome in living cells.

• Single-cell proteomics: PSMB10 antibodies are being adapted for CyTOF and other single-cell protein analysis platforms to study immunoproteasome heterogeneity across cell populations.

• Intravital imaging: Development of non-invasive imaging methods using labeled PSMB10 antibodies or fragments to track immunoproteasome dynamics in living tissues.

• Spatial transcriptomics-proteomics integration: Combining PSMB10 antibody staining with spatial transcriptomics to correlate protein expression with transcriptional programs at single-cell resolution.