PSMB8 Antibody

What is PSMB8 and why is it important for immunological research?

PSMB8, also known as LMP7, PSMB5i, RING10, or Y2, belongs to the peptidase T1B family and encodes the chymotrypsin-like catalytic subunit of the immunoproteasome. It plays essential roles in controlling pathogenic immune responses and may serve as a potential target in autoimmune disorders. The immunoproteasome is crucial for cellular homeostasis, and PSMB8 specifically is required for proper processing of other immunoproteasome subunits (β1i and β2i) . Researchers studying inflammatory conditions, autoimmune disorders, or proteasome function would find PSMB8 particularly relevant as its dysregulation has been linked to autoinflammatory responses and lipodystrophy .

What applications are PSMB8 antibodies validated for?

PSMB8 antibodies have been validated for multiple research applications, including:

These applications allow researchers to detect PSMB8 protein expression, localization, and interactions in various experimental settings .

What tissues and cell types express PSMB8?

PSMB8 expression has been detected in multiple tissues and cell types. Based on the search results, positive Western blot detection has been confirmed in mouse kidney tissue, human kidney tissue, Jurkat cells, and Raji cells . PSMB8 expression has also been observed in skin tissue, with localization in both nuclear and cytoplasmic compartments . Furthermore, B cells express PSMB8, and this expression can be altered in pathological conditions . When designing experiments, researchers should consider these expression patterns to select appropriate positive controls.

How should I optimize PSMB8 antibody usage for immunohistochemistry?

For optimal immunohistochemistry results with PSMB8 antibodies, follow these methodological considerations:

• Antigen retrieval: Use TE buffer pH 9.0 as suggested for optimal epitope exposure. Alternatively, citrate buffer pH 6.0 may be used, but comparative testing is recommended to determine which provides better results for your specific tissue .

• Dilution optimization: Begin with the recommended dilution range (1:50-1:500) but perform a dilution series to determine the optimal concentration for your specific tissue and fixation method .

• Detection system selection: Choose an appropriate detection system based on your microscopy setup and desired sensitivity.

• Positive controls: Include known PSMB8-expressing tissues such as mouse spleen or skin tissue to validate staining specificity .

• Sample preparation: Proper fixation and processing are critical; overfixation may mask epitopes while underfixation can compromise tissue morphology.

What are the key considerations for Western blot detection of PSMB8?

When designing Western blot experiments to detect PSMB8, consider the following technical aspects:

• Molecular weight expectations: The pro-PSMB8 is a 276 amino acid protein with a molecular mass of 30 kDa, but the mature form is approximately 23 kDa due to the cleavage of a 72 amino acid propeptide . This difference is critical for correct interpretation of your results.

• Sample preparation: Complete protein extraction requires appropriate lysis buffers that can solubilize membrane-associated proteins without degrading them.

• Reducing conditions: Ensure consistent reducing conditions in your samples to obtain reliable results.

• Blocking optimization: Determine the most effective blocking agent (BSA vs. non-fat dry milk) for reducing background while maintaining specific signal.

• Positive controls: Include protein lysates from Jurkat cells, Raji cells, or kidney tissue as positive controls .

• Verification strategy: Consider parallel detection of both pro-PSMB8 and mature PSMB8 to assess processing dynamics in your experimental system.

How can I assess immunoproteasome assembly and function using PSMB8 antibodies?

Studying immunoproteasome assembly and function requires sophisticated approaches:

How can PSMB8 antibodies be used to investigate the role of immunoproteasomes in inflammatory conditions?

To study the relationship between PSMB8 and inflammatory responses:

• Cytokine profiling: Analyze the expression of inflammatory cytokines (particularly IL-6) in relation to PSMB8 expression levels using paired antibody detection systems .

• Signaling pathway analysis: Examine p38 phosphorylation and other inflammatory signaling pathways that may be affected by PSMB8 dysfunction .

• Mutation impact assessment: Compare wild-type and mutant PSMB8 expression, stability, and activity in relevant cellular models using pulse-chase experiments and proteasome activity assays .

• Tissue-specific effects: Analyze PSMB8 expression and localization in affected tissues (such as skin) from patients with inflammatory conditions versus healthy controls using immunohistochemistry .

• Knockdown/knockout studies: Use PSMB8 antibodies to confirm efficient knockdown/knockout in model systems before assessing inflammatory phenotypes .

What are common issues with PSMB8 antibody staining and how can they be resolved?

When experiencing problems with PSMB8 antibody applications, consider these methodological solutions:

• High background in immunohistochemistry:

  • Increase blocking time or concentration

  • Optimize antibody dilution (try higher dilutions)

  • Ensure thorough washing between steps

  • Consider using alternative blocking reagents

  • Reduce incubation time with the detection system

• Weak or absent signal:

  • Verify sample preparation and protein extraction

  • Optimize antigen retrieval method (try both TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Decrease antibody dilution

  • Increase incubation time

  • Check if your detection system is working properly with a positive control antibody

• Multiple bands in Western blot:

  • Distinguish between expected pro-PSMB8 (30 kDa) and mature PSMB8 (23 kDa) forms

  • Assess sample degradation

  • Verify antibody specificity through knockout/knockdown controls

  • Consider post-translational modifications

How can I analyze PSMB8 in cells with low expression levels?

When studying PSMB8 in systems with low expression:

• Signal amplification: Consider using tyramide signal amplification (TSA) or other signal-enhancing methods for immunohistochemistry and immunofluorescence.

• Concentrated lysates: Prepare more concentrated protein lysates for Western blotting by increasing the cell number or reducing lysis buffer volume.

• Immunoprecipitation prior to Western blotting: Enrich PSMB8 through immunoprecipitation before Western blot analysis to concentrate the target protein .

• Sensitive detection methods: Use more sensitive chemiluminescent substrates for Western blotting, or consider fluorescent secondary antibodies with digital imaging systems.

• Transfection approaches: Consider transiently overexpressing PSMB8 to establish detection parameters before analyzing endogenous levels.

How can PSMB8 antibodies be used to study the relationship between immunoproteasome function and adipocyte differentiation?

To investigate PSMB8's role in adipocyte biology:

• Expression analysis: Monitor PSMB8 expression during different stages of adipocyte differentiation using Western blotting and immunofluorescence staining .

• siRNA-mediated knockdown: Confirm efficient PSMB8 knockdown using antibody detection before assessing impacts on adipocyte differentiation markers .

• In vivo adipose tissue analysis: Use immunohistochemistry with PSMB8 antibodies to analyze expression patterns in adipose tissue samples from various anatomical locations .

• Quantitative assessment: Combine PSMB8 antibody staining with morphometric analysis to correlate PSMB8 expression levels with adipocyte size, number, and lipid content.

• Co-localization studies: Perform double immunofluorescence staining with PSMB8 antibodies and adipocyte markers to establish spatial relationships within developing and mature adipose tissue.

What experimental approaches can assess the impact of PSMB8 mutations on immunoproteasome function?

To study the effects of PSMB8 mutations on immunoproteasome function:

• Pulse-chase experiments: Use metabolic labeling with [35S]-methionine and cysteine followed by immunoprecipitation with PSMB8 antibodies to assess protein stability of wild-type versus mutant PSMB8 .

• Assembly intermediate analysis: Employ glycerol gradient centrifugation combined with immunoblotting to compare assembly intermediates between wild-type and mutant cells .

• Proteasome activity assays: Compare chymotrypsin-like activities and ubiquitin-independent protein degradation to assess functional consequences of mutations .

• Ubiquitin accumulation: Use immunohistochemistry and Western blotting with ubiquitin antibodies to determine if mutant PSMB8 leads to increased ubiquitinated protein accumulation .

• Inflammatory marker analysis: Correlate PSMB8 expression and mutation status with inflammatory markers such as IL-6 to establish causality in inflammatory conditions .

How might PSMB8 antibodies be utilized in studying autoinflammatory disorders?

For researchers investigating PSMB8 in autoinflammatory conditions:

• Patient sample analysis: Compare PSMB8 expression, localization, and immunoproteasome assembly in tissue samples from patients with autoinflammatory disorders versus healthy controls .

• Cytokine profiling: Correlate PSMB8 expression or mutation status with inflammatory cytokine levels (particularly IL-6) in patient samples .

• Animal model validation: Use PSMB8 antibodies to verify knockout/knockin strategies in animal models of autoinflammatory diseases.

• Therapeutic targeting assessment: Evaluate the effects of potential therapeutic agents targeting immunoproteasome function using PSMB8 antibodies as readouts for target engagement.

• Biomarker development: Explore the potential of PSMB8 expression patterns or post-translational modifications as biomarkers for disease progression or treatment response.

What are the considerations for studying PSMB8 in the context of the complete immunoproteasome complex?

To investigate PSMB8 as part of the integrated immunoproteasome:

• Co-immunoprecipitation studies: Use PSMB8 antibodies to pull down the entire immunoproteasome complex and identify interacting partners through mass spectrometry .

• Stoichiometry analysis: Quantify relative amounts of different immunoproteasome subunits (β1i, β2i, PSMB8) using respective antibodies to assess assembly completeness.

• Functional reconstitution: Verify immunoproteasome reconstitution in cell-free systems using PSMB8 antibodies as quality control markers.

• Comparative analysis: Assess how PSMB8 deficiency or mutation affects other immunoproteasome subunits (β1i, β2i) and their processing .

• Interactome mapping: Combine PSMB8 antibody-based proximity labeling approaches with proteomics to map the extended interaction network of the immunoproteasome.