PSMC5 Antibody, Biotin conjugated

What is PSMC5 and what is its biological significance?

PSMC5 (Proteasome 26S Subunit, ATPase, 5) is a critical component of the 26S proteasome complex, specifically functioning as regulatory subunit 8. This protein acts as an AAA-ATPase within the 19S regulatory particle that controls access to the proteolytic core of the proteasome. PSMC5 participates in the ATP-dependent unfolding of protein substrates and their translocation into the 20S core particle for degradation. Beyond its proteasomal functions, PSMC5 is also known as Thyroid hormone receptor-interacting protein 1 (TRIP1) or p45/SUG, indicating its multifunctional roles in cellular processes that extend beyond protein degradation . The protein plays significant roles in various cellular pathways including transcriptional regulation, DNA repair processes, and cell cycle progression, making it a valuable research target in multiple biological contexts.

What are the key characteristics of the biotin-conjugated PSMC5 antibody?

The biotin-conjugated PSMC5 antibody (Catalog No. ABIN7142239) is a polyclonal antibody raised in rabbits against a specific region (amino acids 253-324) of the human PSMC5 protein . This antibody specifically recognizes the regulatory subunit 8 of the 26S proteasome. The antibody undergoes Protein G purification with >95% purity and is conjugated with biotin, enabling enhanced detection sensitivity through the strong biotin-streptavidin interaction . It shows human reactivity and is validated for ELISA applications. The IgG isotype of this antibody provides reliable performance in immunological techniques, and its polyclonal nature ensures recognition of multiple epitopes within the target region, increasing detection probability under various experimental conditions .

What advantages does biotin conjugation offer in PSMC5 antibody applications?

Biotin conjugation to the PSMC5 antibody provides several methodological advantages in research applications. The biotin-streptavidin system offers one of the strongest non-covalent biological interactions known (Kd ≈ 10^-15 M), which significantly enhances detection sensitivity compared to unconjugated antibodies . This conjugation enables amplified signal generation in detection systems utilizing enzyme-labeled streptavidin conjugates. Additionally, the small size of biotin minimizes interference with antibody-antigen interactions, preserving the specificity and affinity of the original antibody . The versatility of this conjugation allows researchers to employ various detection strategies, including colorimetric, chemiluminescent, or fluorescent methods, by simply changing the streptavidin-reporter conjugate. For multiplex experiments, biotin-conjugated antibodies can be easily integrated with other detection systems, providing flexibility in experimental design without requiring species-specific secondary antibodies .

How can the biotin-conjugated PSMC5 antibody be optimally used in ELISA applications?

For optimal use of biotin-conjugated PSMC5 antibody in ELISA applications, researchers should implement a carefully designed protocol. Begin by coating microplate wells with a capture antibody against PSMC5 (different epitope than the biotin-conjugated antibody) at 1-10 μg/mL in carbonate/bicarbonate buffer (pH 9.6) overnight at 4°C. After washing with PBS-T (PBS + 0.05% Tween-20), block with 2-5% BSA or non-fat milk for 1-2 hours at room temperature. Prepare samples and standards in appropriate dilution buffer and incubate for 1-2 hours. Following washing, apply the biotin-conjugated PSMC5 antibody at a dilution of 1:500-1:5000 (empirical optimization recommended) and incubate for 1-2 hours . Wash thoroughly and incubate with streptavidin-HRP conjugate (typically 1:5000-1:20000) for 30-60 minutes. After final washing, develop with appropriate substrate (TMB for colorimetric detection) and measure absorbance. The biotin-conjugation system significantly enhances detection sensitivity, potentially by 2-4 fold compared to traditional detection methods, making it particularly valuable for detecting low-abundance PSMC5 protein in complex biological samples .

What is the recommended storage and handling protocol to maintain antibody activity?

To maintain optimal activity of the biotin-conjugated PSMC5 antibody, proper storage and handling protocols are essential. Upon receipt, store the antibody at -20°C or -80°C for long-term preservation . When preparing working aliquots, minimize freeze-thaw cycles as repeated freezing and thawing can significantly reduce antibody activity—typically, antibody potency decreases by 10-15% with each freeze-thaw cycle. The storage buffer (50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300) provides stability but should not be diluted until immediately before use . When handling the antibody, maintain a cold chain workflow using an ice bath for temporary storage during experiments. For dilutions, use freshly prepared, sterile buffer preferably containing a carrier protein (0.1-0.5% BSA) to prevent non-specific adsorption to tube walls. It's advisable to prepare single-use aliquots upon first thawing to avoid repeated freeze-thaw cycles. Additionally, centrifuge the antibody vial briefly before opening to collect all liquid at the bottom and ensure proper concentration in each use .

How can the biotin-conjugated PSMC5 antibody be integrated into studies of proteasome activity during cell cycle progression?

To integrate the biotin-conjugated PSMC5 antibody into studies of proteasome activity during cell cycle progression, researchers can employ a multi-methodological approach. First, synchronize cells at specific cell cycle phases using established protocols such as double thymidine block for G1/S boundary, thymidine-nocodazole for mitotic arrest, or serum starvation for G0/G1 . At each phase, extract proteins under non-denaturing conditions to preserve proteasome complex integrity. For direct assessment of PSMC5 dynamics, perform immunoprecipitation using the biotin-conjugated antibody coupled to streptavidin beads, followed by activity assays of the pulled-down proteasome complexes using fluorogenic substrates (e.g., Suc-LLVY-AMC for chymotrypsin-like activity). Alternatively, construct a comprehensive profile by combining flow cytometry for cell cycle analysis with immunofluorescence using the biotin-conjugated PSMC5 antibody and streptavidin-fluorophore conjugates . For studies focusing on mitotic exit, implement the sequential thymidine release/Eg5 inhibitor treatment to arrest cells in prometaphase with monopolar spindles that trigger the SAC (Spindle Assembly Checkpoint), and then release cells through Aurora B kinase inhibition as described in literature protocols . This approach can be particularly valuable when studying ubiquitination patterns during mitotic transitions, where PSMC5 function is critical for proper proteasomal degradation of cell cycle regulators.

How can biotin-conjugated PSMC5 antibodies be used to investigate protein-protein interactions within the 26S proteasome complex?

The biotin-conjugated PSMC5 antibody offers sophisticated approaches for investigating protein-protein interactions within the 26S proteasome complex. Researchers can implement proximity-dependent biotin identification (BioID) by co-immunoprecipitating PSMC5 and its interacting partners using streptavidin-coated magnetic beads. This method leverages the high-affinity biotin-streptavidin interaction (Kd≈10^-15 M) to pull down intact protein complexes with minimal background . For dynamic interaction studies, combine this approach with crosslinking mass spectrometry (XL-MS), where proteins are crosslinked in vivo before isolation with the biotin-conjugated antibody. The biotin tag allows for stringent washing conditions without losing the primary interaction, resulting in cleaner spectra and more reliable identification of transient or weak interactions within the proteasome regulatory particle . Additionally, researchers can develop quantitative interaction maps by performing sequential immunoprecipitations using the biotin-conjugated PSMC5 antibody and antibodies against other proteasome subunits, followed by comparative proteomics analysis. This strategy has revealed that PSMC5 not only interacts with core proteasome components but also with specific transcription factors, supporting its dual role in proteolysis and transcriptional regulation .

What strategies can be employed to study PSMC5 involvement in ubiquitin-dependent protein degradation pathways?

To investigate PSMC5 involvement in ubiquitin-dependent protein degradation pathways, several sophisticated strategies can be implemented using the biotin-conjugated antibody. Researchers can establish in vitro reconstitution systems where purified 26S proteasomes (isolated using the biotin-conjugated PSMC5 antibody) are tested for their ability to degrade ubiquitinated substrates under controlled conditions. This approach allows for precise manipulation of factors such as ATP concentration, which is critical given PSMC5's ATPase activity . For cellular studies, implement a biotin-dependent proximity labeling approach where the PSMC5 antibody is used to identify proteins in spatial proximity to the proteasome during active degradation phases. This can be combined with pulse-chase experiments using biotinylated ubiquitin to track substrate flux through the proteasome system . Advanced time-resolved studies can be designed to capture PSMC5's role during critical cellular transitions, such as mitotic exit, where ubiquitination patterns change dramatically. For instance, experimental designs using sequential thymidine release/Eg5 inhibitor treatment to arrest cells in prometaphase followed by Aurora B kinase inhibition can reveal how PSMC5 contributes to the degradation of cell cycle regulators . These approaches collectively provide a comprehensive view of PSMC5's functional role in connecting ubiquitin recognition to substrate processing within the proteasome machinery.

How can researchers utilize the biotin-conjugated PSMC5 antibody in studies of proteasome dysfunction in disease models?

Utilizing biotin-conjugated PSMC5 antibody in disease model studies provides powerful insights into proteasome dysfunction mechanisms. Researchers can implement multiplex immunofluorescence tissue profiling by combining the biotin-conjugated PSMC5 antibody with antibodies against disease markers, followed by detection with differentially labeled streptavidin conjugates and spectrally distinct secondary antibodies. This approach enables quantitative assessment of PSMC5 expression, localization, and co-localization patterns in relation to pathological features . For functional proteasome assessment in disease contexts, researchers can develop activity-based protein profiling assays by first treating samples with activity-based probes that bind to functional proteasomes, followed by PSMC5 isolation using the biotin-conjugated antibody. This strategy distinguishes between structurally intact but functionally impaired proteasomes versus those with altered PSMC5 expression. In neurodegenerative disease models, where proteasome dysfunction is implicated, the biotin-conjugated antibody can be employed in brain slice immunohistochemistry to map regional variations in PSMC5 expression relative to protein aggregation sites . Additionally, in cancer models where proteasome activity is often dysregulated, researchers can use the antibody to assess whether PSMC5 alterations contribute to resistance mechanisms against proteasome inhibitor therapies, potentially identifying new therapeutic targets or biomarkers.

What are common troubleshooting approaches for optimizing signal-to-noise ratio when using biotin-conjugated PSMC5 antibody?

Optimizing signal-to-noise ratio with biotin-conjugated PSMC5 antibody requires systematic troubleshooting approaches. If experiencing high background, implement a pre-absorption step by incubating the diluted antibody with 5% non-immune serum from the same species as your samples for 1 hour at room temperature before application. This reduces non-specific binding by up to 70% in most applications . Additionally, include a biotin blocking step using commercial biotin blocking kits or sequentially applying avidin (0.1 mg/ml) followed by biotin (0.01 mg/ml) to block endogenous biotin, which can be particularly problematic in tissues rich in biotin such as liver, kidney, and brain . For weak signals, optimize antibody concentration through titration experiments (typically testing dilutions from 1:100 to 1:5000) while simultaneously adjusting incubation times and temperatures. Consider signal amplification systems such as tyramide signal amplification, which can enhance sensitivity by 10-100 fold without increasing background . When performing multiplexed detection, prevent cross-reactivity by ensuring proper blocking between sequential antibody applications and consider using specialized streptavidin conjugates with reduced non-specific binding properties. Finally, if inconsistent results persist, evaluate buffer composition, as the presence of sodium azide can interfere with peroxidase-based detection systems, and detergent concentrations above 0.1% may disrupt some epitopes .

How can researchers verify the specificity of the biotin-conjugated PSMC5 antibody in their experimental system?

To verify specificity of the biotin-conjugated PSMC5 antibody, researchers should implement a multi-level validation approach. Begin with western blot analysis using both recombinant PSMC5 protein and cell/tissue lysates, confirming the detection of a single band at approximately 45 kDa (the expected molecular weight of PSMC5) . Perform peptide competition assays by pre-incubating the antibody with a 5-10 fold molar excess of the immunizing peptide (AA 253-324) before application to samples; specific signal should be significantly reduced or eliminated . For cellular systems, implement genetic approaches by comparing antibody staining patterns between wild-type cells and those with PSMC5 knockdown or knockout; specific staining should decrease proportionally to protein reduction levels. When using the antibody in a new species or application, validate by comparing staining patterns with an alternative validated PSMC5 antibody recognizing a different epitope, such as antibodies targeting AA 127-406 region . Additionally, perform immunoprecipitation followed by mass spectrometry analysis to confirm that the main protein pulled down is indeed PSMC5. For tissue sections, include appropriate control tissues known to express different levels of PSMC5 and compare staining intensity. These comprehensive validation steps ensure that experimental results reflect true PSMC5 biology rather than non-specific interactions .

What strategies can be employed to reduce batch-to-batch variability when using biotin-conjugated polyclonal PSMC5 antibodies in longitudinal studies?

To minimize batch-to-batch variability with biotin-conjugated polyclonal PSMC5 antibodies in longitudinal studies, implement a comprehensive standardization strategy. First, purchase sufficient quantity of a single lot for the entire study duration and create multiple small aliquots (10-20 μl) stored at -80°C to maintain consistent antibody characteristics throughout the research period . For each new batch required, perform side-by-side validation with the original batch using identical samples and protocols across multiple applications to establish a correlation coefficient (aim for r > 0.9) between batches . Develop an internal reference standard by preparing a large batch of positive control lysate or recombinant PSMC5 protein, aliquoted and stored at -80°C, to be included in every experiment as a calibration control. Implement quantitative normalization methods by including standard curves with each experiment and normalizing results to these curves rather than making direct comparisons between experiments performed with different antibody batches . For critical studies, consider developing a pooled antibody approach by mixing small amounts from multiple batches to average out batch-specific variations. Additionally, maintain detailed records of antibody performance metrics for each batch, including signal intensity under standardized conditions, background levels, and optimal working dilutions, allowing for mathematical correction factors to be applied when comparing data generated with different batches .

How should researchers interpret changes in PSMC5 localization detected using the biotin-conjugated antibody?

When interpreting changes in PSMC5 localization detected using the biotin-conjugated antibody, researchers should implement a comprehensive analytical framework. First, establish a baseline localization pattern in control samples, noting that PSMC5 typically exhibits both nuclear and cytoplasmic distribution with enrichment in nucleoplasmic regions and exclusion from nucleoli in most cell types . Quantify localization changes using digital image analysis with nuclear/cytoplasmic ratio calculations, considering a shift of >30% between compartments as biologically significant based on published proteasome dynamics studies. Evaluate the relationship between localization changes and functional outcomes by correlating PSMC5 redistribution with markers of proteasome activity (such as ubiquitinated protein accumulation) and cellular processes (such as cell cycle progression markers) . Consider that transient nuclear-to-cytoplasmic shuttling of PSMC5 often correlates with increased cytoplasmic proteasome activity during stress responses, while sustained nuclear accumulation may indicate transcriptional regulatory functions independent of its proteasomal role . For tissue samples, analyze PSMC5 localization in relation to tissue architecture and pathological features, as altered proteasome distribution has been associated with specific disease states. When observing unusual localization patterns (such as membrane association or punctate structures), perform co-localization studies with markers for relevant organelles or protein aggregates to determine the biological context of the observed redistribution .

What controls should be included when analyzing PSMC5 expression levels using biotin-conjugated antibodies?

When analyzing PSMC5 expression levels using biotin-conjugated antibodies, a comprehensive control strategy is essential for accurate interpretation. Include positive controls consisting of cell lines or tissues known to express PSMC5 at varying levels (e.g., HeLa cells for high expression, differentiated neurons for moderate expression) to establish the dynamic range of detection . Implement negative controls by analyzing samples where PSMC5 has been knocked down via siRNA or CRISPR, with residual expression levels verified by quantitative PCR to serve as calibration standards. Include isotype controls using biotin-conjugated non-specific rabbit IgG at the same concentration as the PSMC5 antibody to distinguish between specific binding and Fc-receptor mediated background . For accurate quantification, incorporate a recombinant PSMC5 standard curve (10-1000 ng/ml) processed identically to experimental samples, allowing conversion of signal intensity to absolute protein quantity. Technical controls should include primary antibody omission, streptavidin-conjugate only, and substrate-only samples to identify sources of background. When analyzing tissues, include biotin blocking controls as endogenous biotin can significantly confound results, particularly in biotin-rich tissues like liver and kidney . Additionally, normalize PSMC5 measurements to multiple housekeeping proteins (not just one) selected based on their stability in your experimental conditions, as standard references like GAPDH or β-actin can vary considerably across treatments and disease states .

How can researchers utilize biotin-conjugated PSMC5 antibodies in combination with super-resolution microscopy techniques?

Integrating biotin-conjugated PSMC5 antibodies with super-resolution microscopy enables unprecedented insights into proteasome spatial organization. For Structured Illumination Microscopy (SIM), researchers should optimize fixation protocols using 4% paraformaldehyde with 0.1% glutaraldehyde to preserve proteasome complexes while enabling antibody penetration, followed by detection using streptavidin conjugated to photostable fluorophores like Alexa Fluor 647 . This approach achieves ~120nm resolution, sufficient to visualize proteasome clusters but not individual complexes. For single-molecule localization microscopy techniques (STORM/PALM), implement a density-based labeling strategy using a mixture of biotin-conjugated PSMC5 antibody and unconjugated PSMC5 antibody (9:1 ratio) to achieve appropriate spatial separation of fluorophores, enabling localization precision of ~20nm . This resolution can reveal previously undetectable proteasome organizational features such as nuclear proteasome clusters associated with transcriptionally active regions. For multi-color super-resolution imaging, combine the biotin-conjugated PSMC5 antibody (detected with streptavidin-Alexa647) with directly labeled antibodies against other proteasome components or interacting proteins, enabling precise colocalization analysis at nanometer-scale resolution . Additionally, researchers can implement expansion microscopy protocols, physically expanding the sample 4-10 fold after immunolabeling with the biotin-conjugated antibody, effectively achieving 25-50nm resolution on standard confocal microscopes. These advanced imaging approaches have revealed that PSMC5-containing proteasomes form distinct functional clusters that dynamically reorganize during cell cycle progression and in response to proteotoxic stress .

What are the considerations for using biotin-conjugated PSMC5 antibodies in studying proteasome dynamics during cellular stress responses?

When studying proteasome dynamics during cellular stress responses with biotin-conjugated PSMC5 antibodies, several methodological considerations are essential. First, implement live-cell imaging approaches by combining the biotin-conjugated antibody with cell-permeable streptavidin-fluorophore conjugates in semi-permeabilized cells, enabling real-time tracking of proteasome redistribution during stress induction with temporal resolution of ~1-5 minutes . Consider the timing of fixation carefully, as proteasome relocalization can occur rapidly (within 15-30 minutes) after stress induction; establish a detailed time-course with points at 0, 15, 30, 60, 120, and 240 minutes post-stress to capture the complete dynamic range . For oxidative stress studies, be aware that strong oxidants can modify the biotin moiety, potentially reducing antibody detection efficiency; implement parallel detection with unconjugated PSMC5 antibodies as controls . When studying heat shock responses, note that temperature elevation may alter epitope accessibility; optimize fixation and antigen retrieval protocols specifically for heat-shocked samples. Develop quantitative metrics for analyzing proteasome dynamics, including measurement of nuclear/cytoplasmic ratios, formation of stress-induced proteasome clusters (quantified by number, size, and intensity), and association with stress granules or processing bodies . Additionally, complement antibody-based detection with functional assays of proteasome activity to distinguish between relocalization events that affect proteasome function versus those that merely reflect passive movement during cellular reorganization. These comprehensive approaches have revealed that PSMC5-containing proteasomes undergo stimulus-specific relocalization patterns that correlate with altered degradation priorities during different stress responses .

How can the biotin-conjugated PSMC5 antibody be utilized in targeted proteomics approaches for biomarker discovery?

The biotin-conjugated PSMC5 antibody offers powerful applications in targeted proteomics for biomarker discovery through several advanced methodological approaches. Implement antibody-based enrichment strategies by using the biotin-conjugated PSMC5 antibody coupled to streptavidin magnetic beads to selectively isolate PSMC5-containing complexes from clinical samples, followed by mass spectrometry analysis to identify differential protein interactions across disease states . This approach has revealed disease-specific interactome signatures with diagnostic potential. Develop a sequential epitope analysis approach where samples are first immunoprecipitated with the biotin-conjugated PSMC5 antibody, followed by analysis with antibodies against post-translational modifications (phosphorylation, ubiquitination, acetylation), enabling identification of modification patterns that correlate with disease progression . For circulating biomarker discovery, implement a proximity extension assay (PEA) combining the biotin-conjugated PSMC5 antibody with DNA oligonucleotide-conjugated antibodies against candidate interacting proteins, allowing ultra-sensitive detection of specific protein complexes in blood or other biofluids . Additionally, develop a PSMC5-centric multiple reaction monitoring (MRM) mass spectrometry panel including specific peptides from PSMC5 and its key interacting partners, enabling absolute quantification of their stoichiometry across patient cohorts . These approaches have identified specific PSMC5 complexes and modification patterns associated with neurodegenerative diseases and certain cancer types, suggesting potential as both diagnostic biomarkers and indicators of therapeutic response to proteasome-targeting drugs .