USP48 Antibody, Biotin conjugated

What is USP48 and what are its main biological functions?

USP48 (Ubiquitin Specific Peptidase 48) is a deubiquitinase enzyme that recognizes and hydrolyzes the peptide bond at the C-terminal glycine of ubiquitin. It plays crucial roles in processing polyubiquitin precursors and ubiquitinated proteins. USP48 is involved in several significant biological processes, including the regulation of NF-kappa-B activation through interactions with RELA and TRAF2, cell cycle progression via deubiquitination of Aurora B/AURKB, and modulation of ciliary and synaptic transport. Additionally, USP48 functions in cytoskeleton organization essential for photoreceptor function and homeostasis by stabilizing retinal degeneration-associated proteins like ARL3 and UNC119. Recent research has also revealed its positive role in pyroptosis by stabilizing gasdermin E/GSDME through removal of 'Lys-48'-linked ubiquitination.

What are the specifications of commercially available USP48 Antibody, Biotin conjugated?

The commercially available USP48 Antibody, Biotin conjugated is a polyclonal antibody raised in rabbits against a recombinant human USP48 protein fragment (amino acids 358-508). This antibody has been purified using Protein G chromatography with a purity of >95%. It is provided in liquid form containing preservative (0.03% Proclin 300) and buffer constituents (50% Glycerol, 0.01M PBS, pH 7.4). The antibody carries the UniProt accession number Q86UV5 and has demonstrated reactivity with human samples in ELISA applications. For optimal performance, storage at -20°C or -80°C is recommended, with precautions to avoid repeated freeze-thaw cycles that could compromise antibody integrity.

How does the biotin conjugation enhance the utility of USP48 antibody in research applications?

Biotin conjugation of the USP48 antibody provides significant advantages for research applications by leveraging the strong non-covalent interaction between biotin and streptavidin/avidin molecules. This modification enables signal amplification through multi-layered detection systems, as each streptavidin molecule can bind four biotin molecules. In immunodetection workflows, this amplification enhances sensitivity, allowing detection of low-abundance USP48 proteins in complex biological samples. Additionally, biotin conjugation facilitates versatile experimental designs as the antibody can be used with various detection systems (fluorescent, enzymatic, or chemiluminescent streptavidin conjugates) without requiring species-specific secondary antibodies. This versatility makes the biotin-conjugated USP48 antibody particularly valuable for multiplex immunoassays where several antibodies must be used simultaneously without cross-reactivity issues.

What are the recommended applications for USP48 Antibody, Biotin conjugated in research settings?

The USP48 Antibody, Biotin conjugated has been validated primarily for ELISA applications when studying human samples. For complementary approaches, researchers should note that non-conjugated versions of USP48 antibodies have demonstrated utility in additional techniques including immunoprecipitation (IP), Western blotting (WB), and immunocytochemistry/immunofluorescence (ICC/IF). When designing experiments with the biotin-conjugated variant, optimization of antibody concentration is essential for each specific application. For ELISA, typical starting dilutions range from 1:500 to 1:2000, though this may require adjustment based on sample type and detection method. The biotin conjugation makes this antibody particularly suitable for avidin-biotin complex (ABC) detection systems and for multi-color immunofluorescence studies where differentiation of multiple antigens is required.

How should researchers design validation experiments for USP48 Antibody, Biotin conjugated?

A comprehensive validation strategy for USP48 Antibody, Biotin conjugated should include multiple complementary approaches. First, researchers should perform specificity testing using positive controls (tissues or cell lines known to express USP48, such as HeLa or 293T cells) and negative controls (USP48 knockout or knockdown samples). Cross-reactivity assessment is crucial, particularly when working with samples from different species or when analyzing tissues with similar deubiquitinases. A titration experiment determining optimal antibody concentration should be conducted to establish the working range where signal-to-noise ratio is maximized. For confirming signal specificity, competitive inhibition assays using recombinant USP48 protein can be valuable. Additionally, researchers should verify results using alternative detection methods or independent antibodies targeting different epitopes of USP48. All validation experiments should include proper controls for the biotin-streptavidin detection system, including controls for endogenous biotin and non-specific binding of streptavidin conjugates.

What is the recommended protocol for using USP48 Antibody, Biotin conjugated in ELISA?

This protocol should be optimized for specific experimental conditions. Critical considerations include sample preparation, antibody dilution optimization, incubation times and temperatures, and appropriate positive and negative controls to ensure reliable results.

What are common troubleshooting strategies when working with USP48 Antibody, Biotin conjugated?

When encountering challenges with USP48 Antibody, Biotin conjugated experiments, systematic troubleshooting approaches should be implemented. For high background signals, optimization of blocking conditions is critical—consider extending blocking time or using alternative blocking reagents such as casein or commercial blocking solutions. If signal intensity is weak, several strategies can be employed: increasing antibody concentration, extending incubation time, or using more sensitive detection methods. For non-specific binding issues, increasing washing stringency (more washes or higher detergent concentration) often helps. Additionally, pre-adsorption of the antibody with irrelevant proteins can reduce cross-reactivity. If inconsistent results occur between replicates, standardizing sample handling procedures and ensuring uniform reagent preparation becomes crucial. When troubleshooting specificity concerns, validating with knockout/knockdown controls or comparing with non-conjugated antibodies targeting the same epitope can provide valuable insights into the nature of the problem.

How should researchers interpret discrepancies between predicted and observed molecular weights in Western blot applications?

Discrepancies between predicted and observed molecular weights of USP48 in Western blot applications are not uncommon and require careful interpretation. The predicted molecular weight of USP48 is approximately 119 kDa, while observed bands may appear at approximately 145 kDa, as reported in validation studies. These variations can result from several factors: post-translational modifications (particularly ubiquitination, phosphorylation, or glycosylation), alternative splicing generating different isoforms, conformational characteristics that affect protein migration, or the presence of strongly bound interaction partners that resist denaturation conditions. When encountering such discrepancies, researchers should implement additional validation approaches, including mass spectrometry analysis, immunoprecipitation followed by Western blotting with alternative antibodies, or verification with genetic knockdown/knockout samples. Comparing band patterns across different cell types or tissues can also provide insights into tissue-specific modifications or isoform expression patterns.

What controls should be included when using USP48 Antibody, Biotin conjugated in experimental workflows?

A comprehensive control strategy for experiments using USP48 Antibody, Biotin conjugated should include multiple elements to ensure reliable interpretation of results:

• Positive controls: Samples known to express USP48 (e.g., HeLa or 293T cell lysates)

• Negative controls: USP48 knockout/knockdown samples or tissues known not to express USP48

• Specificity controls: Pre-incubation of antibody with recombinant USP48 protein to block specific binding

• Biotin-specific controls: Samples treated with unconjugated streptavidin to identify endogenous biotin

• Secondary reagent controls: Samples incubated with detection reagent only (no primary antibody)

• Loading/normalization controls: Housekeeping proteins or total protein stains for Western blots

• Isotype controls: Irrelevant biotin-conjugated IgG antibodies to assess non-specific binding

• Technical replicates: Multiple samples processed identically to evaluate reproducibility

• Cross-validation: Parallel experiments using alternative USP48 antibodies targeting different epitopes

How can USP48 Antibody, Biotin conjugated be utilized in studies investigating deubiquitination mechanisms?

USP48 Antibody, Biotin conjugated offers sophisticated applications for investigating deubiquitination mechanisms. Researchers can employ this antibody in co-immunoprecipitation experiments to identify protein complexes associated with USP48, revealing potential substrates and regulatory partners in specific cellular contexts. By combining the antibody with proximity ligation assays (PLA), researchers can visualize and quantify direct interactions between USP48 and candidate substrates in situ. For temporal dynamics studies, the biotin-conjugated antibody can be used in pulse-chase experiments with cycloheximide to monitor USP48 protein stability or in combination with ubiquitin antibodies to track deubiquitination kinetics of specific substrates. The antibody can also facilitate the investigation of USP48's role in specific cellular compartments through subcellular fractionation followed by immunoblotting, or through super-resolution microscopy for precise localization studies. For substrate identification, researchers can perform comparative ubiquitinomic analyses in systems with normal versus depleted USP48, using the antibody to confirm USP48 knockdown efficiency.

What methodological approaches can be used to study the role of USP48 in NF-κB signaling pathways?

To investigate USP48's role in NF-κB signaling pathways, researchers can implement several sophisticated methodological approaches using USP48 Antibody, Biotin conjugated. Chromatin immunoprecipitation (ChIP) assays can be performed using the antibody to determine if USP48 is recruited to NF-κB-regulated gene promoters. For protein-protein interaction studies, sequential immunoprecipitation (first with anti-RELA antibodies followed by USP48 Antibody) can identify specific complexes containing both proteins. Researchers can combine CRISPR-Cas9 mediated USP48 gene editing with reporter assays measuring NF-κB transcriptional activity to establish causal relationships. To understand the dynamics of the interaction, live-cell imaging using fluorescently-tagged RELA combined with fixed-cell immunofluorescence using the biotin-conjugated USP48 antibody allows correlation of USP48 expression with RELA nuclear translocation. For H. pylori infection studies, time-course experiments measuring nuclear RELA levels (using subcellular fractionation followed by Western blotting) in conjunction with USP48 activity assays can reveal the temporal relationship between USP48 activity and NF-κB signaling during infection progression.

How can researchers integrate USP48 Antibody, Biotin conjugated into multi-omics experimental designs?

Integration of USP48 Antibody, Biotin conjugated into multi-omics experimental designs enables comprehensive characterization of USP48's functional networks. For proteomics integration, immunoprecipitation using the antibody followed by mass spectrometry can identify the USP48 interactome, which can then be correlated with transcriptomic data from USP48-depleted cells to identify genes directly and indirectly regulated by USP48 activity. Researchers can perform ChIP-seq using the biotin-conjugated antibody to map USP48 genomic binding sites, which can be integrated with RNA-seq data to establish direct transcriptional targets. For spatial proteomics approaches, multiplexed immunofluorescence combining the USP48 antibody with antibodies against various cellular markers can reveal compartment-specific functions when integrated with subcellular proteomics data. To understand post-translational modification networks, researchers can combine USP48 immunoprecipitation with ubiquitinome analysis to identify the spectrum of substrates deubiquitinated by USP48. Additionally, correlation of USP48 expression or localization with metabolomic profiles in various cellular states can reveal its influence on cellular metabolism. These integrated approaches provide a systems-level understanding of USP48 function beyond what individual omics approaches can achieve.

What are the key differences between biotin-conjugated and unconjugated USP48 antibodies for different experimental applications?

The biotin-conjugated and unconjugated USP48 antibodies present distinct advantages and limitations across various experimental applications. For Western blotting, unconjugated antibodies typically offer more straightforward protocols with direct secondary antibody detection, while biotin-conjugated versions require an additional streptavidin-enzyme conjugate step but potentially provide enhanced sensitivity through signal amplification. In immunocytochemistry/immunofluorescence applications, biotin-conjugated antibodies excel in multiplexing scenarios where multiple primary antibodies from the same host species need to be differentiated, and they offer flexibility in detection modalities (fluorescent, chromogenic, or chemiluminescent). For flow cytometry, biotin conjugation enables signal amplification and multicolor analysis through various streptavidin-fluorophore conjugates, particularly valuable for detecting low-abundance USP48. In chromatin immunoprecipitation, unconjugated antibodies are generally preferred for direct immunoprecipitation, while biotin-conjugated versions are advantageous for sequential ChIP experiments. For ELISA applications, biotin-conjugated antibodies offer superior sensitivity and flexibility, allowing various detection enzymes to be introduced via streptavidin conjugates without changing the primary antibody.

How does the choice of epitope region affect the performance of USP48 antibodies in different experimental contexts?

The choice of epitope region significantly impacts USP48 antibody performance across experimental contexts. Antibodies targeting the middle region of USP48 (such as those recognizing amino acids 358-508) typically provide robust detection in Western blotting and ELISA, as these regions may be more accessible in partially denatured proteins. Conversely, antibodies targeting the catalytic domain might be more effective for functional studies, potentially inhibiting USP48 enzymatic activity. For protein interaction studies, antibodies against non-catalytic regions are preferable to avoid disrupting protein-protein interfaces. Epitope accessibility varies dramatically between applications: for Western blotting where proteins are denatured, internal epitopes perform well, while for immunoprecipitation or immunofluorescence where native conformations are maintained, surface-exposed epitopes are superior. Additionally, epitope choice affects cross-reactivity with USP48 homologs (like USP31) and alternative splice variants. When studying USP48 in complex with specific substrates, epitope selection should avoid regions involved in substrate binding to prevent epitope masking. Researchers must carefully consider these factors when selecting antibodies for specific experimental questions.

What methodological approaches can researchers use to validate the specificity of USP48 Antibody, Biotin conjugated in ubiquitin pathway studies?

Validation of USP48 Antibody, Biotin conjugated specificity in ubiquitin pathway studies requires rigorous methodological approaches. Researchers should implement genetic validation using CRISPR-Cas9 knockout or siRNA knockdown of USP48, confirming antibody specificity by demonstrating signal reduction in depleted samples. Orthogonal validation comparing results with alternative antibodies targeting different USP48 epitopes provides confirmation of target specificity. For recombinant protein validation, researchers can perform competitive binding assays where pre-incubation of the antibody with purified recombinant USP48 should abolish specific signals. Domain-specific validation using expression constructs of individual USP48 domains can precisely map the antibody's recognition site. To assess cross-reactivity with other deubiquitinating enzymes, parallel detection of closely related DUBs (particularly USP31) is essential. Substrate-specific validation can be achieved by monitoring changes in ubiquitination status of known USP48 substrates (such as Aurora B/AURKB, RELA, or GSDME) following USP48 depletion or overexpression. Additionally, mass spectrometry validation comparing immunoprecipitated proteins identified by the antibody with the USP48 protein sequence provides definitive identification. These complementary approaches collectively establish antibody specificity within the complex context of ubiquitin pathway studies.