PSMC4 Antibody, FITC conjugated

What is PSMC4 and what role does it play in cellular processes?

PSMC4 (proteasome 26S subunit, ATPase, 4) is a critical component of the 26S proteasome complex involved in ATP-dependent degradation of ubiquitinated proteins. Also known as TBP7, S6, and MIP224, PSMC4 belongs to the AAA ATPase family and participates in protein quality control mechanisms essential for cellular homeostasis . The protein has a calculated molecular weight of 47 kDa, though it typically appears between 43-50 kDa in Western blot applications, likely due to post-translational modifications . PSMC4 functions within the regulatory particle of the 26S proteasome, contributing to substrate recognition, unfolding, and translocation into the catalytic core.

How does FITC conjugation affect PSMC4 antibody applications?

FITC (Fluorescein Isothiocyanate) conjugation provides direct fluorescent labeling of the PSMC4 antibody, eliminating the need for secondary antibody incubation steps in immunofluorescence and flow cytometry applications. This conjugation results in green fluorescence (emission ~520 nm) when excited with blue light (~495 nm). While unconjugated PSMC4 antibodies require multi-step detection protocols, FITC-conjugated variants offer several methodological advantages:

• Simplified experimental workflows with fewer incubation and washing steps

• Reduced background signal by eliminating potential cross-reactivity from secondary antibodies

• Enhanced capacity for multiplexing with antibodies raised in the same host species

• Direct visualization capabilities in flow cytometry and live-cell imaging applications

What species reactivity is documented for available PSMC4 antibodies?

According to validation data, commercially available PSMC4 antibodies demonstrate confirmed reactivity with human, mouse, and rat samples . Positive Western blot detection has been specifically verified in mouse and rat brain tissues . Immunohistochemistry applications have been validated with human colon cancer tissue . Immunofluorescence has been successfully performed in human cell lines including SH-SY5Y and HepG2 cells . Additional cross-reactivity with pig, zebrafish, bovine, horse, sheep, rabbit, dog, and Xenopus species has been predicted through sequence homology analysis but would require experimental verification .

What are the optimal application parameters for PSMC4 antibody in immunofluorescence studies?

When designing immunofluorescence experiments with FITC-conjugated PSMC4 antibody, researchers should consider the following optimized parameters:

For optimal results in co-localization studies, ensure filter sets are properly configured to minimize bleed-through between fluorescence channels, and consider sequential rather than simultaneous image acquisition when using multiple fluorophores.

How should samples be prepared for Western blot detection of PSMC4?

For Western blot applications targeting PSMC4, the following protocol elements are critical for successful detection:

• Sample preparation: Lyse cells or tissues in RIPA buffer containing protease inhibitors to prevent degradation of PSMC4 protein

• Protein quantification: Load 20-50 μg of total protein per lane for optimal detection

• Gel selection: Use 10-12% SDS-PAGE gels for optimal resolution around the 43-50 kDa range

• Transfer conditions: Semi-dry or wet transfer at 100V for 60-90 minutes using PVDF membrane

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature

• Antibody dilution: 1:500-1:2000 dilution range is recommended, with 1:1000 as a starting point

• Detection method: When using FITC-conjugated antibodies, visualization requires a fluorescence imaging system rather than chemiluminescence

Expected molecular weight observation should be between 43-50 kDa based on validation data from multiple sources . For quantitative analysis, normalization to housekeeping proteins such as GAPDH or β-actin is essential.

What are the critical considerations for immunohistochemical detection of PSMC4 in tissue samples?

Successful immunohistochemical detection of PSMC4 in tissue sections requires careful attention to antigen retrieval and antibody concentration:

• Tissue preparation: Formalin-fixed, paraffin-embedded (FFPE) sections cut at 4-6 μm thickness

• Deparaffinization: Standard xylene and graded ethanol series

• Antigen retrieval: TE buffer pH 9.0 is specifically recommended, with citrate buffer pH 6.0 as an alternative

• Blocking: 10% normal serum and 1% BSA in PBS for 1 hour at room temperature

• Primary antibody: Dilution range of 1:50-1:500, with optimization required for specific tissue types

• Incubation time: Overnight at 4°C for maximum sensitivity

• Detection system: For FITC-conjugated antibodies, direct fluorescence visualization; for unconjugated, appropriate HRP-conjugated secondary antibody with DAB substrate

Positive control tissues should include human colon cancer tissue, which has been validated for PSMC4 expression . Appropriate negative controls (primary antibody omission) are essential for distinguishing specific staining from autofluorescence, particularly when using FITC conjugates.

How can researchers address common technical issues with FITC-conjugated PSMC4 antibodies?

When working with FITC-conjugated PSMC4 antibodies, researchers frequently encounter several technical challenges that can be systematically addressed:

For FITC-specific concerns, researchers should be particularly mindful of the fluorophore's sensitivity to pH, with optimal fluorescence occurring at slightly alkaline conditions (pH 8.0). Maintaining proper storage conditions (4°C, protected from light) for the conjugated antibody is also crucial for preserving activity.

What validation methods should be employed to confirm PSMC4 antibody specificity?

Rigorous validation of PSMC4 antibody specificity is essential for generating reliable experimental data. Researchers should implement multiple complementary approaches:

• Genetic controls: Compare staining patterns between wild-type samples and those with PSMC4 knockdown/knockout (siRNA or CRISPR-Cas9)

• Peptide competition: Pre-incubate antibody with immunizing peptide before application to verify signal elimination

• Multi-method verification: Confirm expression patterns using alternative techniques (qPCR, mass spectrometry)

• Cross-antibody validation: Compare staining patterns using multiple antibodies targeting different PSMC4 epitopes

• Positive controls: Include established PSMC4-expressing cell lines like SH-SY5Y or HepG2

• Western blot correlation: Confirm antibody detects a single band of appropriate molecular weight (43-50 kDa)

• Isotype controls: Use isotype-matched immunoglobulins to assess non-specific binding

These validation steps should be systematically documented and included in publications to establish antibody reliability for the specific experimental conditions employed.

How can PSMC4 antibody signal be distinguished from proteasome complex background?

Distinguishing specific PSMC4 signal from general proteasomal staining requires sophisticated experimental approaches:

• Comparative subunit analysis: Perform parallel staining with antibodies against different proteasome subunits to establish unique PSMC4 localization patterns

• Super-resolution microscopy: Employ techniques like STORM or STED to resolve individual proteasome complexes beyond the diffraction limit

• Proximity ligation assays: Use PLA technology to specifically detect PSMC4 interaction with other proteasome components

• Subcellular fractionation: Isolate distinct cellular compartments before antibody application to reduce complexity

• Quantitative co-localization: Calculate Pearson's correlation coefficients between PSMC4 and other proteasome markers to identify unique distribution patterns

• Proteasome disassembly conditions: Compare PSMC4 staining patterns under conditions that promote proteasome disassembly

These approaches allow researchers to distinguish between PSMC4 staining representing assembled proteasome complexes versus free PSMC4 subunits, providing insight into proteasome assembly dynamics.

How can FITC-conjugated PSMC4 antibodies be applied to study proteasome dynamics in neurodegenerative disease models?

Proteasome dysfunction is implicated in numerous neurodegenerative disorders, making PSMC4 a valuable research target. FITC-conjugated PSMC4 antibodies enable sophisticated experimental approaches in this context:

• Comparative neuropathology: Quantify PSMC4 expression and distribution changes in postmortem brain tissues from patients with Alzheimer's, Parkinson's, or Huntington's disease compared to age-matched controls

• Protein aggregation interactions: Perform dual-label immunofluorescence to assess PSMC4 co-localization with disease-specific protein aggregates (e.g., Aβ plaques, α-synuclein inclusions, polyQ aggregates)

• Stress response dynamics: Track real-time changes in PSMC4 distribution following proteasome inhibition or oxidative stress in primary neuron cultures

• Cell-type specific vulnerabilities: Combine FITC-PSMC4 antibody with neuronal, astrocytic, and microglial markers to determine cell-type specific proteasome disruptions

• Patient-derived models: Analyze PSMC4 expression in iPSC-derived neurons from patients with genetic forms of neurodegeneration

• Therapeutic interventions: Assess PSMC4 distribution changes in response to proteasome-modulating compounds

These applications benefit from the direct fluorescent labeling provided by FITC conjugation, particularly in multi-label immunofluorescence studies where antibody species cross-reactivity can be limiting.

What insights can be gained from studying PSMC4's potential role in viral infection mechanisms?

While the search results focus primarily on PSMB4's antiviral functions , the proteasome system broadly impacts viral infection cycles, suggesting potential PSMC4 involvement deserving investigation:

• Viral protein interactions: Investigate whether PSMC4, like PSMB4, interacts with viral proteins to modulate their stability or function

• Ubiquitination patterns: Analyze how viral infection alters K63-linked ubiquitination patterns of proteins associated with PSMC4

• Innate immune signaling: Examine PSMC4's potential role in regulating NF-κB signaling activation during viral challenges

• Autolysosomal pathway: Determine whether PSMC4 contributes to the degradation of viral components through LC3 interaction similar to PSMB4's documented activity

• ATPase functionality: Investigate whether PSMC4's ATPase activity is manipulated by viral factors to alter proteasome function

• Proteasome redistribution: Use FITC-conjugated PSMC4 antibodies to track proteasome redistribution during different stages of viral infection

These research directions could reveal previously uncharacterized host defense mechanisms or viral evasion strategies, particularly given that PSMB4 has demonstrated antiviral activities through both direct viral protein interactions and immune signaling enhancement .

What methodological approaches can optimize PSMC4 detection in challenging experimental conditions?

Advanced researchers often encounter complex experimental conditions requiring specialized approaches for PSMC4 detection:

• Low abundance detection: Employ tyramide signal amplification (TSA) systems compatible with FITC to enhance signal without increasing background

• Multiplexed imaging: Utilize spectral unmixing algorithms to separate FITC signal from potentially overlapping fluorophores or autofluorescence

• Live-cell applications: Consider antibody fragments (Fab) conjugated with FITC for improved cellular penetration in live-cell studies

• Tissue clearing techniques: Combine FITC-conjugated PSMC4 antibody with CLARITY or iDISCO tissue clearing for whole-organ imaging at depth

• High-content screening: Develop automated image analysis workflows to quantify PSMC4 expression across large sample sets

• Super-resolution compatibility: Pair FITC-conjugated antibodies with appropriate buffer systems for optimal performance in STORM or STED microscopy

• Quantitative approaches: Implement standards for converting fluorescence intensity to absolute protein concentration through calibrated imaging

These methodological refinements can significantly enhance detection sensitivity and specificity, particularly in challenging samples like fixed archival tissues or systems with high autofluorescence like brain and liver tissues.

How should researchers analyze and interpret changes in PSMC4 localization patterns?

Quantitative analysis of PSMC4 localization requires systematic approaches to distinguish biologically meaningful changes from technical variability:

• Standardized image acquisition: Maintain consistent exposure settings, laser power, and detector gain across experimental groups

• Subcellular compartment analysis: Employ nuclear/cytoplasmic masks to quantify compartment-specific PSMC4 distribution

• Co-localization metrics: Calculate Manders' overlap coefficient or Pearson's correlation coefficient for PSMC4 with organelle markers

• Pattern recognition: Apply artificial intelligence algorithms to identify subtle changes in distribution patterns

• Time-series analysis: For dynamic studies, implement trajectory analysis to track PSMC4 movement between compartments

• Statistical approaches: Apply appropriate statistical tests for distribution comparisons, accounting for cell-to-cell variability

• Visualization methods: Present data as heat maps or 3D surface plots to represent complex distribution patterns effectively

What experimental controls are essential for studying PSMC4 in protein degradation pathways?

When investigating PSMC4's role in protein degradation, several critical controls must be implemented:

• Proteasome inhibitor controls: Include MG132 treatment groups to distinguish proteasome-dependent from proteasome-independent degradation mechanisms

• Lysosomal pathway controls: Incorporate bafilomycin A1 or chloroquine treatment to block lysosomal degradation, which can compensate for proteasome inhibition

• Ubiquitination profiling: Monitor changes in both K48-linked (proteasomal) and K63-linked (often non-degradative) ubiquitination patterns

• ATPase activity controls: Include ATP depletion conditions to distinguish energy-dependent from energy-independent PSMC4 functions

• Substrate specificity controls: Compare degradation patterns of known proteasome substrates with varying characteristics (short-lived vs. long-lived proteins)

• PSMC4 manipulation validation: Confirm knockdown/overexpression efficiency through multiple methods (Western blot, qPCR)

• Alternative ATPase subunit controls: Compare PSMC4 manipulation with alterations to other proteasomal ATPases (PSMC1-6) to identify unique functions

These controls enable researchers to distinguish PSMC4-specific effects from general proteasome functions and provide context for interpreting experimental outcomes in protein degradation studies.

How might PSMC4 research contribute to understanding cancer therapy resistance mechanisms?

The proteasome is a validated therapeutic target in multiple cancer types, with PSMC4 potentially playing critical roles in therapy response:

• Inhibitor binding sites: Investigate whether structural differences in PSMC4 across cancer types influence proteasome inhibitor efficacy

• Compensatory mechanisms: Examine whether PSMC4 upregulation compensates for inhibition of catalytic proteasome subunits

• Cancer-specific interactions: Identify cancer-specific protein interactions with PSMC4 that could be targeted therapeutically

• Biomarker potential: Evaluate PSMC4 expression patterns as potential predictive biomarkers for proteasome inhibitor therapy

• Combination approaches: Test whether targeted PSMC4 modulation sensitizes resistant cancer cells to existing therapeutics

• Non-proteolytic functions: Explore potential non-canonical functions of PSMC4 in cancer cell signaling networks

These research directions could reveal new strategies for overcoming resistance to proteasome-targeting therapies in various cancer contexts through PSMC4-specific approaches.

What emerging technologies might enhance PSMC4 functional studies beyond antibody-based detection?

While antibody-based detection remains valuable, emerging technologies offer complementary approaches to study PSMC4 biology:

• Proximity labeling proteomics: Apply BioID or APEX2 fusions to PSMC4 to map its protein interaction network in living cells

• CRISPR-based imaging: Utilize dCas9-fluorophore fusions for endogenous PSMC4 visualization without antibodies

• Single-molecule tracking: Apply Halo-Tag or SNAP-Tag fusions for real-time tracking of individual PSMC4 molecules

• Intrabodies: Develop intracellularly expressed antibody fragments against PSMC4 for live-cell applications

• nanobody detection: Employ camelid-derived single-domain antibodies conjugated with FITC for improved penetration and reduced size

• Mass spectrometry imaging: Apply spatial proteomics approaches to map PSMC4 distribution in tissues without antibodies

• Optical biosensors: Develop FRET-based sensors to monitor PSMC4 conformational changes during proteasome activation

These emerging approaches complement traditional antibody-based detection methods and enable novel experimental designs for understanding PSMC4 dynamics and function in increasingly physiological contexts.