MBTPS1 Antibody

What is MBTPS1 and what is its significance in cellular processes?

MBTPS1 (also known as S1P, SEDKF, SKI-1, membrane-bound transcription factor site-1 protease, endopeptidase S1P, and PCSK8) is a serine protease belonging to the Peptidase S8 protein family . In humans, the canonical protein consists of 1052 amino acid residues with a molecular mass of approximately 117.7 kDa . MBTPS1 is primarily localized in the endoplasmic reticulum (ER) and Golgi apparatus and is widely expressed across numerous tissue types .

The enzyme plays a critical role in cellular function by cleaving substrate proteins after hydrophobic or small residues, provided that arginine or lysine is in position P4 . This proteolytic activity is essential for activating various transcription factors and regulatory proteins involved in lipid metabolism, ER stress response, and other cellular pathways.

Which model organisms have MBTPS1 orthologs suitable for research?

MBTPS1 is highly conserved across species, making it amenable to comparative research approaches. Orthologs have been identified and characterized in multiple model organisms including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken . This conservation allows researchers to study MBTPS1 function in various experimental systems, providing insights that may be applicable across species or highlighting species-specific differences in MBTPS1 regulation and function.

What are the known substrates of MBTPS1 and how can they be utilized in experimental designs?

Several key substrates of MBTPS1 have been identified, including SREBF1/SREBP1, SREBF2/SREBP2, BDNF, GNPTAB, ATF6, ATF6B, and FAM20C . These substrates are involved in diverse cellular processes including:

• Lipid metabolism regulation (SREBF1/SREBP1, SREBF2/SREBP2)

• Neurotrophin signaling (BDNF)

• Lysosomal enzyme targeting (GNPTAB)

• Unfolded protein response (ATF6, ATF6B)

• Biomineralization and phosphorylation of secreted proteins (FAM20C)

When designing experiments, researchers can target these substrates to study the downstream effects of MBTPS1 activity or inhibition. For instance, monitoring the processing of SREBP transcription factors can serve as a readout for MBTPS1 activity in lipid metabolism studies, while examining ATF6 cleavage can provide insights into the role of MBTPS1 during ER stress responses.

Which applications are most effective for MBTPS1 antibody-based detection?

Based on research publications and technical specifications, several applications have proven effective for MBTPS1 antibody-based detection:

• Western Blot (WB): This is the most widely used application for MBTPS1 antibodies, allowing for size-based detection and semi-quantitative analysis of MBTPS1 protein levels . Western blotting is particularly useful for distinguishing between the precursor and processed forms of MBTPS1.

• Immunofluorescence (IF): Many MBTPS1 antibodies have been validated for IF applications, enabling researchers to visualize the subcellular localization of MBTPS1 within the ER and Golgi compartments .

• Enzyme-Linked Immunosorbent Assay (ELISA): ELISA provides a quantitative approach for measuring MBTPS1 levels in various sample types and is supported by numerous commercially available antibodies .

• Immunohistochemistry (IHC): IHC allows for the detection of MBTPS1 in tissue sections, providing insights into expression patterns across different cell types within complex tissues .

• Immunoprecipitation (IP): Some antibodies have been validated for IP applications, facilitating the isolation of MBTPS1 and its interacting partners for further analysis.

What are the critical parameters for optimizing Western blot protocols for MBTPS1 detection?

When optimizing Western blot protocols for MBTPS1 detection, several critical parameters should be considered:

• Sample preparation: Given MBTPS1's membrane localization, complete solubilization is essential. Use detergent-containing lysis buffers (e.g., RIPA or NP-40 supplemented with protease inhibitors) to effectively extract MBTPS1 from ER and Golgi membranes.

• Protein denaturation: MBTPS1 is a large protein (117.7 kDa) with multiple domains . Complete denaturation is crucial for accurate size determination. Sample heating at 95°C for 5-10 minutes in SDS-containing buffer is recommended, but protein aggregation should be monitored.

• Gel percentage: Due to MBTPS1's size, lower percentage gels (6-8%) or gradient gels are optimal for proper resolution.

• Transfer conditions: Extended transfer times or semi-dry transfer systems optimized for large proteins may be necessary for efficient transfer of MBTPS1 to membranes.

• Antibody selection: Choose antibodies with validated Western blot applications that recognize epitopes maintained under denaturing conditions . Consider the specific MBTPS1 domain targeted by the antibody and whether it will detect both precursor and processed forms.

• Blocking conditions: Optimize blocking solutions (typically 3-5% BSA or non-fat dry milk) to minimize background while maintaining specific signal.

How can MBTPS1 antibodies be effectively used in immunofluorescence and immunohistochemistry studies?

For effective use of MBTPS1 antibodies in IF and IHC applications:

• Fixation method: Choose appropriate fixation methods that preserve MBTPS1 epitopes while maintaining cellular architecture. Paraformaldehyde (4%) is often suitable, but methanol fixation may better preserve some epitopes.

• Permeabilization: Since MBTPS1 is mainly localized to intracellular membranes (ER and Golgi) , effective permeabilization is crucial. Triton X-100 (0.1-0.5%) or saponin (0.1-0.3%) are commonly used permeabilization agents.

• Antigen retrieval: For formalin-fixed paraffin-embedded tissues, heat-induced epitope retrieval (citrate buffer pH 6.0 or EDTA buffer pH 9.0) may be necessary to expose MBTPS1 epitopes.

• Antibody validation: Confirm antibody specificity using appropriate positive and negative controls. Tissues or cells with known MBTPS1 expression levels can serve as positive controls, while siRNA-mediated knockdown samples can function as negative controls.

• Co-localization markers: Include markers for ER (e.g., calnexin, PDI) and Golgi (e.g., GM130, TGN46) to confirm the expected subcellular localization of MBTPS1 and distinguish it from other cellular compartments.

• Signal amplification: For tissues with lower MBTPS1 expression, consider using signal amplification methods like tyramide signal amplification or higher sensitivity detection systems.

How can MBTPS1 antibodies be utilized to study the enzyme's activation and trafficking dynamics?

Studying MBTPS1 activation and trafficking requires sophisticated experimental approaches using specific antibodies:

• Epitope-specific antibodies: Utilize antibodies recognizing different domains of MBTPS1 to track its maturation through proteolytic processing. Antibodies targeting the N-terminal prodomain versus the catalytic domain can differentiate between inactive precursor and mature active forms.

• Pulse-chase experiments: Combine metabolic labeling with immunoprecipitation using MBTPS1 antibodies to track the kinetics of MBTPS1 maturation and trafficking through the secretory pathway.

• Live-cell imaging: For dynamic studies, express fluorescently tagged MBTPS1 and validate its localization using antibody-based immunofluorescence as a control. This approach allows real-time tracking of MBTPS1 movement between cellular compartments.

• Subcellular fractionation: Use differential centrifugation to isolate ER and Golgi fractions, followed by Western blotting with MBTPS1 antibodies to quantify the distribution of MBTPS1 across these compartments under various experimental conditions.

• Proximity labeling: Combine MBTPS1 antibodies with techniques like BioID or APEX2 to identify proteins in close proximity to MBTPS1, revealing potential interaction partners involved in its trafficking and activation.

What approaches can be used to study MBTPS1 substrate specificity using antibody-based methods?

Several antibody-based approaches can be employed to investigate MBTPS1 substrate specificity:

• Cleavage site-specific antibodies: Develop or obtain antibodies that specifically recognize the intact substrate (pre-cleavage) or the cleaved products. These can be used to monitor MBTPS1 activity toward specific substrates like SREBP1/2, ATF6, or other known targets .

• In vitro cleavage assays: Recombinant substrates can be incubated with immunoprecipitated MBTPS1 (using specific antibodies), followed by analysis of cleavage products using cleavage site-specific antibodies or other detection methods.

• Cellular assays with substrate reporters: Express fluorescently tagged MBTPS1 substrates and monitor their processing in response to various stimuli. Validate these findings using MBTPS1 antibodies to correlate substrate cleavage with MBTPS1 activation or localization.

• Proximity ligation assay (PLA): Use MBTPS1 antibodies in combination with substrate-specific antibodies to detect and quantify direct interactions between MBTPS1 and its substrates in situ.

• Phospho-specific antibodies: Since phosphorylation may regulate MBTPS1 activity or substrate recognition, phospho-specific antibodies can help elucidate regulatory mechanisms affecting substrate specificity.

How can researchers effectively distinguish between MBTPS1 and other related proteases using antibody-based approaches?

Distinguishing MBTPS1 from related proteases requires careful antibody selection and experimental design:

• Epitope selection: Choose antibodies targeting unique regions of MBTPS1 that are not conserved in related proteases, particularly those in the Peptidase S8 family .

• Validation with recombinant proteins: Test antibody cross-reactivity against purified recombinant MBTPS1 and related proteases to ensure specificity.

• Knockout/knockdown controls: Validate antibody specificity using MBTPS1 knockout or knockdown models. Signal absence in these models confirms antibody specificity.

• Immunoprecipitation followed by mass spectrometry: Perform IP with MBTPS1 antibodies followed by mass spectrometry to confirm the identity of the precipitated protein and detect any cross-reactivity with related proteases.

• Competitive binding assays: Pre-incubate antibodies with recombinant MBTPS1 before application to samples to demonstrate that signal reduction is due to specific binding.

• Comparative expression analysis: Use MBTPS1 antibodies alongside antibodies for related proteases to create expression profiles across tissues or cell types, highlighting the unique distribution pattern of MBTPS1.

What are common causes of false positives or false negatives in MBTPS1 antibody experiments and how can they be addressed?

Several factors can lead to false results when working with MBTPS1 antibodies:

Causes of False Positives:

• Cross-reactivity: Some antibodies may recognize proteins with similar epitopes to MBTPS1. Solution: Validate antibody specificity using knockout/knockdown controls and competing peptide blocking experiments.

• Non-specific binding: Particularly in tissues with high proteolytic activity or complex protein mixtures. Solution: Optimize blocking conditions, increase washing stringency, and verify results with alternative antibodies targeting different MBTPS1 epitopes.

• Overcrowded Western blots: MBTPS1's size (117.7 kDa) places it in a region where many other abundant proteins migrate. Solution: Use gradient gels for better separation and include appropriate loading controls.

Causes of False Negatives:

• Epitope masking: The target epitope may be inaccessible due to protein folding, post-translational modifications, or protein-protein interactions. Solution: Test multiple antibodies targeting different regions of MBTPS1 or adjust sample preparation methods.

• Inefficient extraction: MBTPS1's membrane localization can make it difficult to extract. Solution: Use more stringent lysis buffers containing appropriate detergents for membrane protein extraction.

• Protein degradation: MBTPS1, as a protease, may be sensitive to degradation during sample preparation. Solution: Use fresh samples, maintain cold conditions throughout processing, and include protease inhibitors in all buffers.

• Insufficient antigen retrieval in IHC/IF: Solution: Optimize antigen retrieval methods by testing different buffers, pH values, and heating protocols.

How can researchers verify the specificity of MBTPS1 antibody signals in their experimental system?

Verifying MBTPS1 antibody specificity should involve multiple complementary approaches:

• Genetic validation: Use CRISPR/Cas9 knockout, siRNA knockdown, or shRNA models to confirm that signal reduction correlates with reduced MBTPS1 expression.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to block specific binding sites before application to samples. Specific signals should be significantly reduced or eliminated.

• Multiple antibodies: Use several antibodies targeting different MBTPS1 epitopes. Concordant results across different antibodies increase confidence in specificity.

• Expected molecular weight: Confirm that the detected protein migrates at the expected molecular weight (approximately 117.7 kDa for the full-length protein) , with potential additional bands representing known processing forms.

• Expected subcellular localization: Verify that the detected protein localizes to the ER and Golgi apparatus , as expected for MBTPS1, using co-localization with known organelle markers.

• Recombinant protein control: Include purified recombinant MBTPS1 as a positive control in Western blot experiments to confirm the correct band size.

• Functional validation: Correlate antibody-detected MBTPS1 with functional readouts, such as substrate (SREBP1/2, ATF6) cleavage .

What approaches can help resolve discrepancies in MBTPS1 expression data when different antibodies yield conflicting results?

When faced with discrepancies between different MBTPS1 antibodies:

• Epitope mapping: Determine the exact epitopes recognized by each antibody. Discrepancies may be explained by differential recognition of MBTPS1 isoforms, processed forms, or post-translationally modified variants.

• Cross-validation with non-antibody methods: Use orthogonal approaches such as RT-qPCR for mRNA expression, mass spectrometry for protein identification, or activity-based protein profiling for functional protein detection.

• Cell type and context considerations: Different antibodies may perform differently depending on cell type, fixation method, or experimental conditions. Systematically test each antibody across various experimental conditions.

• Protein-protein interactions: Some antibodies may be affected by MBTPS1's interactions with other proteins. Use denaturing conditions in Western blots or appropriate sample preparation for IF/IHC to minimize these effects.

• Sequential probing: For Western blots, strip and reprobe membranes with different antibodies to compare band patterns on identical samples.

• Literature cross-reference: Compare your findings with published literature to identify patterns in antibody performance and reported discrepancies.

• Manufacturer consultation: Contact antibody manufacturers for technical support regarding known issues, lot-to-lot variability, or application-specific optimizations.

How are MBTPS1 antibodies being used to study the role of this protease in pathological conditions?

Recent research has employed MBTPS1 antibodies to investigate its role in various pathological conditions:

• Metabolic disorders: MBTPS1 antibodies have been used to study altered SREBP processing in conditions like hyperlipidemia, insulin resistance, and hepatic steatosis. By monitoring MBTPS1 expression and activation, researchers can correlate its activity with dysregulated lipid metabolism.

• Neurodegenerative diseases: Given MBTPS1's role in processing BDNF and its involvement in ER stress responses , antibody-based studies have examined its contribution to conditions like Alzheimer's disease, where ER stress is a key pathogenic mechanism.

• Cancer biology: MBTPS1 antibodies help investigate how altered MBTPS1 activity might contribute to cancer cell survival under stress conditions, particularly through activation of the ATF6 branch of the unfolded protein response .

• Infectious diseases: Several viruses hijack MBTPS1 for their life cycle. Antibody-based detection of MBTPS1 interaction with viral proteins provides insights into viral pathogenesis mechanisms.

• Rare genetic disorders: Mutations in MBTPS1 substrates like GNPTAB cause conditions such as mucolipidosis. MBTPS1 antibodies help characterize how these substrates are processed in healthy versus diseased states .

What are emerging methodologies incorporating MBTPS1 antibodies for high-throughput or single-cell analyses?

Emerging methodologies combining MBTPS1 antibodies with advanced techniques include:

• Single-cell proteomics: Integration of MBTPS1 antibodies with mass cytometry (CyTOF) or microfluidic-based single-cell Western blotting to analyze MBTPS1 expression and activation at the single-cell level, revealing cell-to-cell variability within tissues.

• Spatial proteomics: Combination of MBTPS1 antibodies with technologies like multiplexed ion beam imaging (MIBI) or co-detection by indexing (CODEX) to map MBTPS1 expression and activity within tissue architecture with subcellular resolution.

• Automated high-content imaging: Implementation of MBTPS1 antibody-based immunofluorescence in high-content screening platforms to assess how various compounds affect MBTPS1 localization, processing, or activity in large-scale experiments.

• Phospho-proteomics: Integration of MBTPS1 immunoprecipitation with phospho-proteomics to map regulatory phosphorylation sites on MBTPS1 and how they change under various conditions.

• Antibody-based biosensors: Development of FRET-based or split-fluorescent protein biosensors incorporating MBTPS1 antibody fragments to monitor MBTPS1 activity or conformation changes in living cells.

How can researchers integrate MBTPS1 antibody applications with other 'omics approaches for comprehensive analysis?

Integration of MBTPS1 antibody applications with other 'omics approaches enables more comprehensive analysis:

• Proteomics integration: Combine MBTPS1 immunoprecipitation with mass spectrometry to identify interaction partners and how these interactions change under different physiological or pathological conditions.

• Transcriptomics correlation: Pair MBTPS1 protein expression data from antibody-based methods with RNA-seq data to identify discrepancies between transcript and protein levels, potentially revealing post-transcriptional regulation mechanisms.

• ChIP-seq applications: For transcription factor substrates of MBTPS1 (like SREBPs) , combine MBTPS1 antibody detection of substrate processing with ChIP-seq of the cleaved, active transcription factors to connect MBTPS1 activity with downstream transcriptional changes.

• Metabolomics integration: Correlate MBTPS1 activity (measured by antibody detection of processed substrates) with metabolomic profiles, particularly lipid profiles, given MBTPS1's role in SREBP processing and lipid metabolism .

• Systems biology modeling: Incorporate quantitative data from MBTPS1 antibody experiments into computational models of relevant signaling pathways, particularly ER stress responses and lipid metabolism regulation.

• Multi-'omics data visualization: Develop integrated visualization approaches that combine MBTPS1 antibody-derived protein data with transcriptomic, metabolomic, and other 'omics data to identify patterns and relationships across multiple molecular levels.

What criteria should researchers consider when selecting an MBTPS1 antibody for specific applications?

Several key criteria should guide MBTPS1 antibody selection:

For complex studies, researchers may need multiple antibodies targeting different epitopes to comprehensively analyze MBTPS1 biology.

How do various fixation and sample preparation methods affect MBTPS1 antibody performance?

Fixation and sample preparation significantly impact MBTPS1 antibody performance:

How can researchers design experiments to study MBTPS1 activity under ER stress conditions using antibody-based approaches?

To study MBTPS1 activity during ER stress:

• Stress induction monitoring: Use MBTPS1 antibodies in combination with ER stress markers (BiP/GRP78, CHOP, phospho-eIF2α) to correlate MBTPS1 activation with ER stress induction using pharmacological inducers (thapsigargin, tunicamycin, DTT) or physiological stressors.

• Substrate processing assays: Monitor ATF6 and SREBP processing using specific antibodies that distinguish between full-length and cleaved forms. Compare processing kinetics under normal versus ER stress conditions.

• Subcellular redistribution: Use immunofluorescence with MBTPS1 antibodies to track potential relocalization between ER and Golgi during stress responses, co-labeling with compartment markers.

• Activation-state specific detection: If available, use conformation-specific antibodies that preferentially recognize the active form of MBTPS1 to directly monitor activation during ER stress.

• Co-immunoprecipitation: Use MBTPS1 antibodies for IP followed by immunoblotting for interaction partners to identify stress-dependent protein associations.

• Pulse-chase analysis: Combine metabolic labeling with immunoprecipitation using MBTPS1 antibodies to track changes in MBTPS1 turnover and processing rates during ER stress.

• Sequential tissue or cell sampling: In disease models with progressive ER stress, use MBTPS1 antibodies to monitor expression and activity changes over time, correlating with disease progression.

What methodological approaches can help researchers study post-translational modifications of MBTPS1 using antibody-based techniques?

Studying MBTPS1 post-translational modifications requires specialized approaches:

• Modification-specific antibodies: When available, use antibodies specifically recognizing phosphorylated, glycosylated, or otherwise modified MBTPS1. These can be used in Western blotting, IF, or IP applications.

• Enzyme treatments: Prior to immunodetection with MBTPS1 antibodies, treat samples with phosphatases, glycosidases, or other enzymes that remove specific modifications. Comparing modified versus demodified samples reveals the presence and extent of modifications.

• 2D gel electrophoresis: Separate MBTPS1 based on both molecular weight and isoelectric point before immunodetection to resolve differentially modified forms.

• IP-MS approach: Immunoprecipitate MBTPS1 using validated antibodies followed by mass spectrometry analysis to identify and characterize post-translational modifications.

• Phos-tag gels: For phosphorylation studies, use Phos-tag acrylamide gels to separate phosphorylated from non-phosphorylated MBTPS1 forms, followed by immunodetection with MBTPS1 antibodies.

• Sequential immunoprecipitation: First IP with modification-specific antibodies (e.g., anti-phosphotyrosine) followed by Western blotting with MBTPS1 antibodies, or vice versa.

• In vitro modification assays: Treat immunoprecipitated MBTPS1 with kinases, glycosyltransferases, or other modifying enzymes in vitro, then analyze with appropriate antibodies to study modification sites and effects.

What emerging technologies might enhance the specificity and utility of MBTPS1 antibodies in research applications?

Several emerging technologies promise to enhance MBTPS1 antibody applications:

• Nanobodies and single-domain antibodies: These smaller antibody fragments offer improved access to sterically hindered epitopes within the transmembrane regions or catalytic pocket of MBTPS1.

• Recombinant antibody engineering: Custom-designed recombinant antibodies with improved specificity, reduced cross-reactivity, and optimized binding properties for specific MBTPS1 domains or conformational states.

• Aptamer-based detection: Development of DNA or RNA aptamers as alternatives or complements to traditional antibodies, potentially offering advantages in certain applications.

• CRISPR knock-in tags: Endogenous tagging of MBTPS1 using CRISPR/Cas9 to facilitate detection with highly specific anti-tag antibodies, avoiding limitations of direct MBTPS1 antibodies.

• Proximity labeling enhancements: Integration of MBTPS1 antibodies with advanced proximity labeling techniques (TurboID, APEX2) for improved mapping of the MBTPS1 interactome under various conditions.

• Proteolytic activity-based probes: Development of probes that become detectable upon cleavage by active MBTPS1, allowing direct visualization or quantification of enzymatic activity rather than just protein levels.

• Super-resolution microscopy optimization: Design of fluorophore-conjugated MBTPS1 antibodies specifically optimized for super-resolution techniques to study nanoscale localization and dynamics.