PSMC4 Antibody, Biotin conjugated

What is PSMC4 and what biological role does the protein play?

PSMC4 (Proteasome 26S Subunit, ATPase, 4) functions as a regulatory subunit 6B within the 26S proteasome complex. It is also known by alternative names including MIP224 and TBP7 (Tat-binding protein 7). The protein serves as an AAA-ATPase subunit (RPT3) within the 19S regulatory particle of the 26S proteasome, which is crucial for ATP-dependent degradation of ubiquitinated proteins in eukaryotic cells. PSMC4 specifically contributes to the recognition, unfolding, and translocation of proteins destined for degradation into the 20S core particle of the proteasome .

What are the basic characteristics of PSMC4 Antibody, Biotin conjugated?

PSMC4 Antibody, Biotin conjugated is a polyclonal antibody raised in rabbits against specific epitopes of the human PSMC4 protein. The antibody recognizes amino acid positions 112-181 of the regulatory subunit 6B. It undergoes Protein G purification with >95% purity and is conjugated with biotin, which facilitates detection through avidin/streptavidin systems. The antibody is typically supplied in liquid form in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative .

What is the significance of biotin conjugation for this antibody?

Biotin conjugation provides significant advantages in immunodetection techniques by leveraging the exceptional affinity between biotin and streptavidin/avidin. This conjugation enables versatile detection strategies without requiring direct enzyme labeling of the primary antibody. The biotin tag allows for signal amplification through multiple binding sites on streptavidin molecules, enhancing detection sensitivity in techniques like ELISA, immunohistochemistry, and other assays where signal strength is critical. The small size of biotin means it typically does not interfere with the antibody's antigen-binding capabilities .

What are the validated applications for PSMC4 Antibody, Biotin conjugated?

The primary validated application for this biotin-conjugated PSMC4 antibody is Enzyme-Linked Immunosorbent Assay (ELISA). While the biotin-conjugated version is specifically optimized for ELISA, other forms of PSMC4 antibodies (non-biotin conjugated) have demonstrated effectiveness in Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), and Immunocytochemistry (ICC) applications. When selecting this antibody, researchers should confirm that biotin conjugation is appropriate for their specific experimental needs, as different detection methods may require different antibody conjugations .

How should PSMC4 Antibody, Biotin conjugated be stored and handled to maintain activity?

For optimal preservation of antibody activity, PSMC4 Antibody, Biotin conjugated should be stored at -20°C or -80°C immediately upon receipt. Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and diminished antibody performance. When handling the antibody, it is recommended to aliquot it into single-use volumes before freezing to minimize freeze-thaw cycles. Working dilutions should be prepared fresh before use and stored at 4°C for short-term applications (less than a week). The stabilizing buffer containing 50% glycerol helps maintain antibody integrity during the freeze-thaw process .

What are the recommended protocols for using PSMC4 Antibody, Biotin conjugated in ELISA?

For optimal ELISA performance with PSMC4 Antibody, Biotin conjugated:

• Coat the plate with target antigen (typically recombinant PSMC4 or samples containing PSMC4)

• Block with appropriate blocking buffer (typically 5% BSA or milk in PBST)

• Apply primary antibody at optimized dilution (starting recommendations: 1:1000-1:5000)

• Wash thoroughly with PBST (PBS + 0.05% Tween-20)

• Apply streptavidin-HRP conjugate (1:5000-1:10000 dilution)

• Wash thoroughly

• Develop with appropriate substrate (TMB for HRP)

• Read absorbance

Optimization may be required for specific experimental conditions, including determination of ideal antibody concentration through titration experiments .

What is the species reactivity profile of this antibody?

The PSMC4 Antibody, Biotin conjugated has been specifically validated for reactivity with human PSMC4 protein. The immunogen used for antibody generation was a recombinant human 26S proteasome regulatory subunit 6B protein fragment (amino acids 112-181). While this particular biotin-conjugated formulation has only been validated for human reactivity, it's worth noting that other non-conjugated PSMC4 antibody variants may show cross-reactivity with additional species including mouse and rat PSMC4, depending on sequence conservation in the epitope region .

How can I validate the specificity of PSMC4 Antibody, Biotin conjugated in my experimental system?

To validate specificity in your experimental system:

• Positive Controls: Use tissues or cell lines known to express PSMC4 (many human cell lines express this proteasome component)

• Negative Controls: Include samples where primary antibody is omitted but all other detection reagents are applied

• Blocking Peptide Competition: Pre-incubate the antibody with excess immunizing peptide (amino acids 112-181 of human PSMC4) before application to samples

• Knockdown Validation: Compare staining between normal samples and those with PSMC4 knockdown (siRNA)

• Western Blot Correlation: Confirm that band pattern in Western blot matches expected molecular weight (~47 kDa)

• Multi-antibody Comparison: Compare staining patterns with other antibodies targeting different PSMC4 epitopes

These validation approaches ensure that the observed signals genuinely represent PSMC4 rather than non-specific binding .

How can PSMC4 Antibody, Biotin conjugated be used to study proteasome complex assembly and function?

For investigating proteasome complex assembly and function:

• Co-immunoprecipitation Analysis: Use the biotin-conjugated antibody with streptavidin beads to pull down PSMC4 and analyze co-precipitating proteasome components

• Proximity Ligation Assay (PLA): Combine this antibody with antibodies against other proteasome subunits to visualize and quantify protein-protein interactions within the complex

• FACS Analysis: After cell permeabilization, use this antibody to quantify PSMC4 levels under different cellular conditions

• Chromatin Immunoprecipitation (ChIP): When PSMC4 is involved in transcriptional regulation, use this antibody to identify associated DNA regions

• Proteasome Activity Correlation: Correlate PSMC4 expression levels (detected by this antibody) with measured proteasome activity in cell or tissue samples

These approaches leverage the biotin-conjugation to facilitate sensitive detection in complex experimental systems .

What considerations should be made when using this antibody in pathological tissue samples?

When using PSMC4 Antibody, Biotin conjugated in pathological samples:

• Endogenous Biotin Blocking: Pathological tissues (especially liver, kidney, and some tumors) may contain high levels of endogenous biotin, requiring specific blocking steps (using avidin/biotin blocking kits)

• Antigen Retrieval Optimization: Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has been validated for PSMC4 detection in paraffin-embedded sections

• Multiple Control Tissues: Include both normal and pathological controls to establish baseline expression patterns

• Background Reduction: Optimize blocking with 10% goat serum to minimize non-specific binding

• Signal Amplification Considerations: For tissues with low PSMC4 expression, consider tyramide signal amplification systems

Immunohistochemistry data shows successful PSMC4 detection in human glioblastoma, lung adenocarcinoma, rectum adenocarcinoma, and urothelial carcinoma tissues using appropriate antigen retrieval and detection methods .

How can I use this antibody to evaluate PSMC4 involvement in cancer biology research?

For cancer biology applications:

• Expression Profiling: Compare PSMC4 expression levels between normal and cancerous tissues using immunohistochemistry and tissue microarrays

• Subcellular Localization Studies: Use immunofluorescence to track PSMC4 localization changes during cancer progression

• Correlation Analysis: Correlate PSMC4 expression with clinical parameters, treatment response, and patient outcomes

• Drug Response Studies: Monitor PSMC4 levels before and after proteasome inhibitor treatment

• Cancer Stem Cell Analysis: Investigate PSMC4 in cancer stem cell populations using dual staining approaches

The antibody has been successfully used to detect PSMC4 in various cancer tissue samples including glioblastoma, lung adenocarcinoma, rectum adenocarcinoma, and urothelial carcinoma, indicating its utility in cancer research applications .

What are common problems encountered with PSMC4 Antibody, Biotin conjugated and how can they be resolved?

Methodical troubleshooting focusing on these parameters can help resolve most detection issues encountered with this antibody .

How can I optimize dual immunofluorescence experiments with PSMC4 Antibody, Biotin conjugated and other antibodies?

For successful dual immunofluorescence experiments:

• Sequential Detection Protocol:

  • Apply and detect the non-biotin conjugated primary antibody first

  • Apply PSMC4 Antibody, Biotin conjugated

  • Use streptavidin conjugated to a different fluorophore than the first detection system

• Cross-Reactivity Prevention:

  • Select primary antibodies from different host species when possible

  • Use highly cross-adsorbed secondary reagents

  • Consider using Fab fragments for blocking when antibodies are from the same species

• Signal Optimization:

  • Balance signal intensities through antibody dilution optimization

  • Select fluorophores with minimal spectral overlap

  • Include appropriate single-stained controls for each detection channel

• Imaging Considerations:

  • Acquire images sequentially rather than simultaneously

  • Implement proper controls for channel bleed-through

  • Use spectral unmixing when necessary

Example: Successful double immunofluorescence has been demonstrated using rabbit anti-PSMC4 antibody alongside mouse anti-Beta Tubulin antibody (M01857-3) in U2OS cells, with detection using Cy3-conjugated anti-rabbit IgG and DyLight488-conjugated anti-mouse IgG .

How might PSMC4 Antibody, Biotin conjugated be utilized in studying proteasome dysfunction in neurodegenerative diseases?

PSMC4 Antibody, Biotin conjugated offers several methodological approaches for investigating proteasome dysfunction in neurodegenerative conditions:

• Comparative Expression Analysis: Quantify PSMC4 levels in affected versus unaffected brain regions using immunohistochemistry with biotin-streptavidin detection systems

• Co-localization Studies: Examine spatial relationships between PSMC4 and disease-specific protein aggregates (e.g., amyloid plaques, tau tangles, α-synuclein inclusions)

• Sequential Protein Extraction Analysis: Compare PSMC4 distribution in soluble versus insoluble protein fractions from disease models

• Age-dependent Expression Profiling: Track PSMC4 levels across disease progression in longitudinal studies

• Response to Therapeutic Interventions: Monitor PSMC4 expression changes following treatments targeting protein homeostasis

The biotin conjugation provides signal amplification advantages that may be particularly valuable for detecting subtle changes in proteasome component expression or localization in neuronal populations .

What are the considerations for using PSMC4 Antibody, Biotin conjugated in super-resolution microscopy applications?

For super-resolution microscopy applications with PSMC4 Antibody, Biotin conjugated:

• Signal Conversion Strategies:

  • Utilize small, monovalent streptavidin conjugates with appropriate fluorophores for STORM/PALM

  • Consider quantum dot-conjugated streptavidin for STED microscopy

  • Employ site-specific secondary labeling techniques for DNA-PAINT

• Sample Preparation Optimization:

  • Implement specialized fixation protocols that preserve ultrastructure

  • Minimize fixation-induced autofluorescence

  • Utilize appropriate mounting media with antifade properties

• Controls and Validation:

  • Perform correlative imaging with conventional methods

  • Validate localization patterns with antibodies targeting other proteasome components

  • Include specifically designed spatial calibration standards

• Data Analysis Considerations:

  • Apply cluster analysis algorithms to quantify nanoscale distribution patterns

  • Implement distance measurement between PSMC4 and other proteasome components

  • Consider 3D reconstruction to fully capture spatial organization

The small size of the biotin-streptavidin detection system provides advantages for achieving the resolution necessary to study proteasome complex architecture at the nanoscale level .

How does PSMC4 Antibody, Biotin conjugated compare with other detection methods for studying proteasome function?

Selection should be based on experimental requirements, sample type, and research questions. The biotin-conjugated antibody provides excellent sensitivity for fixed samples but may not be optimal for all experimental designs .

What criteria should guide the selection between different commercial sources of PSMC4 Antibody, Biotin conjugated?

When selecting between commercial sources of PSMC4 Antibody, Biotin conjugated, consider:

• Validation Data Comprehensiveness:

  • Extent of application validation (beyond manufacturer claims)

  • Availability of positive and negative control images

  • Publication record using the specific antibody clone

• Technical Specifications:

  • Epitope region targeted (amino acids 112-181 is common)

  • Clonality (polyclonal offers multiple epitope recognition)

  • Purification method (Protein G purification >95% purity is standard)

  • Conjugation chemistry and biotin:antibody ratio

• Lot-to-Lot Consistency:

  • Manufacturer's quality control measures

  • Availability of lot-specific validation data

  • Consistency in production methods

• Support and Documentation:

  • Detailed protocols for specific applications

  • Technical support availability

  • Certificate of analysis completeness

• Storage and Stability:

  • Buffer composition (50% glycerol with preservative is standard)

  • Shelf-life information

  • Shipping conditions

Careful evaluation of these criteria helps ensure selection of the most appropriate reagent for specific research needs .