MBTPS2 Antibody

What is MBTPS2 and why is it significant in research applications?

MBTPS2 (membrane-bound transcription factor peptidase, site 2) is a critical intramembrane protease that functions in the regulated intramembrane proteolysis pathway. It plays essential roles in cholesterol homeostasis, ER stress response, and sterol regulatory element-binding protein (SREBP) activation. The protein is approximately 57.4 kilodaltons in mass and has been identified across multiple species including human, mouse, rat, and other mammalian models .

Research significance stems from MBTPS2's involvement in numerous pathological conditions. Mutations in MBTPS2 have been linked to several X-linked disorders including ichthyosis follicularis with atrichia and photophobia (IFAP) syndrome, keratosis follicularis spinulosa decalvans (KFSD), and Olmsted syndrome, making it a valuable target for both basic research and potential therapeutic development .

What are the key differences between polyclonal and monoclonal MBTPS2 antibodies?

Polyclonal MBTPS2 antibodies (such as the rabbit polyclonal antibody from Proteintech) recognize multiple epitopes on the MBTPS2 protein, offering high sensitivity for applications like ELISA and immunofluorescence with reactivity across human, mouse, and rat samples . These antibodies typically provide robust signals even when protein expression is low but may exhibit batch-to-batch variation.

Monoclonal MBTPS2 antibodies (such as the 1A3 clone) recognize a single epitope, providing excellent specificity for applications requiring fine discrimination. These are particularly valuable for applications like Western blot, where cross-reactivity must be minimized . For example, Cell Signaling Technology's monoclonal antibody has been cited in 25 publications, demonstrating its reliability in research settings .

Methodologically, researchers should select polyclonal antibodies when broader epitope recognition is beneficial (detecting denatured proteins in WB or formalin-fixed tissues) and monoclonal antibodies when absolute specificity is required (distinguishing closely related proteins or isoforms).

What validation methods confirm MBTPS2 antibody specificity?

Proper validation of MBTPS2 antibodies requires multiple complementary approaches:

• Western blot validation: Successful detection of bands at the expected molecular weights (36-57 kDa depending on isoform/modification state) in relevant tissue/cell lysates .

• Positive and negative controls: Testing antibodies on samples with confirmed MBTPS2 expression versus knockdown/knockout samples or tissues known to lack expression.

• Immunoprecipitation followed by mass spectrometry: This technique confirms that the precipitated protein is indeed MBTPS2.

• Cross-species reactivity testing: Confirmation of reactivity across human, mouse, and rat samples as specified in product documentation .

• Application-specific validation: For immunohistochemistry applications, demonstration of expected subcellular localization patterns (primarily ER/Golgi membrane localization for MBTPS2).

When evaluating published work utilizing MBTPS2 antibodies, researchers should look for citations demonstrating successful use in their application of interest, such as the 25 publications citing Cell Signaling Technology's antibody .

How should researchers optimize MBTPS2 detection in membrane protein fractionation experiments?

MBTPS2 is an intramembrane protease primarily localized to the ER and Golgi membranes, requiring specialized techniques for optimal detection:

• Membrane protein extraction protocol:

  • Use gentle detergents like 0.5-1% NP-40 or 1% Triton X-100 in initial extraction

  • Include protease inhibitors (PMSF, leupeptin, aprotinin) to prevent degradation

  • Maintain samples at 4°C throughout processing to preserve membrane integrity

• Subcellular fractionation optimization:

  • Employ differential centrifugation (10,000g for crude membranes, 100,000g for microsomes)

  • Use discontinuous sucrose gradients (0.25M-2.0M) for separating ER/Golgi fractions

  • Verify fraction purity using established markers (calnexin for ER, GM130 for Golgi)

• Western blot considerations:

  • Avoid excessive sample heating (limit to 70°C for 10 minutes) to prevent aggregation

  • Use 7.5-10% gels for optimal resolution of the 57.4 kDa MBTPS2 protein

  • Include positive controls from tissues known to express MBTPS2 (liver, brain)

• Detection optimization:

  • Extended transfer times (90-120 minutes) improve transfer efficiency of membrane proteins

  • BSA (3-5%) is preferred over milk for blocking to reduce background

  • Overnight primary antibody incubation at 4°C often yields superior results

For researchers employing immunofluorescence microscopy, permeabilization conditions significantly impact MBTPS2 detection. Digitonin (0.005%) selectively permeabilizes plasma membranes while preserving ER/Golgi structures, often yielding clearer results than Triton X-100 for transmembrane proteins.

What are the critical considerations when using MBTPS2 antibodies for co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) of MBTPS2 with its interaction partners requires careful experimental design:

• Antibody selection considerations:

  • Choose antibodies validated for immunoprecipitation (IP) applications

  • The Santa Cruz sc-293341 antibody has been specifically validated for IP applications

  • Consider epitope location - avoid antibodies targeting regions involved in protein-protein interactions

• Lysis buffer optimization:

  • Use CHAPS (1%) or digitonin (1%) detergents to maintain native protein conformations

  • Include phosphatase inhibitors if studying phosphorylation-dependent interactions

  • Adjust salt concentration (150-300mM NaCl) based on interaction strength

• Experimental controls:

  • Include IgG control IP to identify non-specific binding

  • Perform reverse Co-IP (IP with antibody against interacting protein) to confirm interactions

  • Include input samples (5-10% of lysate used for IP) for quantitative comparison

• Protocol modifications for membrane proteins:

  • Pre-clear lysates extensively (2-3 rounds) to reduce non-specific membrane protein binding

  • Consider crosslinking approaches for transient interactions

  • Extended incubation times (overnight at 4°C) may improve IP efficiency

When interpreting Co-IP data, researchers should be particularly attentive to the detergent conditions, as they significantly impact which MBTPS2 interactions are preserved. SREBP-MBTPS2 interactions are particularly sensitive to extraction conditions and may require specialized approaches like proximity ligation assays as complementary techniques.

How can researchers effectively use MBTPS2 antibodies in studying ER stress responses?

MBTPS2 plays a critical role in ER stress response through its involvement in SREBP and ATF6 processing. Effective experimental approaches include:

• Time-course analysis protocol:

  • Induce ER stress with tunicamycin (2-5 μg/ml), thapsigargin (100-300 nM), or DTT (1-2 mM)

  • Collect samples at multiple timepoints (0, 2, 4, 8, 16, 24 hours)

  • Process for both protein (Western blot) and RNA (qPCR) analysis

  • Monitor MBTPS2 activity through detection of cleaved ATF6 fragments

• Subcellular translocation methodology:

  • Use immunofluorescence with anti-MBTPS2 antibodies optimized for IF applications

  • Co-stain with compartment markers (calnexin for ER, GM130 for Golgi)

  • Employ confocal microscopy for precise localization

  • Quantify colocalization using Pearson's or Mander's coefficients

• Proteolytic activity assessment:

  • Implement substrate cleavage assays using SREBP or ATF6 reporters

  • Compare activity under normal versus stress conditions

  • Include controls with MBTPS2 inhibitors (e.g., Nelfinavir)

• Genetic manipulation approaches:

  • Use siRNA knockdown to assess necessity of MBTPS2 in stress responses

  • For rescue experiments, consider expressing MBTPS2 variants identified in human diseases

  • Validate antibody specificity in knockdown/knockout systems

When designing ER stress experiments, researchers should consider that MBTPS2 activity may vary by cell type and stress condition. Liver-derived cells often show robust MBTPS2-dependent responses due to their high SREBP activity, making them excellent model systems for initial characterization studies.

What are the optimal fixation and permeabilization conditions for MBTPS2 immunostaining?

MBTPS2's transmembrane nature requires careful optimization of fixation and permeabilization for immunofluorescence and immunohistochemistry:

• Fixation protocol optimization:

  Fixative	Concentration	Duration	Best For
  Paraformaldehyde	2-4%	10-15 min	General IF applications
  Methanol	100%	5-10 min at -20°C	Preserving membrane structures
  Paraformaldehyde/Glutaraldehyde	4%/0.1%	15 min	Electron microscopy

• Permeabilization considerations:

  Agent	Concentration	Duration	Notes
  Triton X-100	0.1-0.2%	5-10 min	Strong permeabilization, may disrupt membranes
  Saponin	0.1%	10-15 min	Reversible, gentler on membranes
  Digitonin	0.005-0.01%	5-10 min	Selective for plasma membrane, preserves ER/Golgi

• Antigen retrieval methods:

  • Heat-mediated retrieval: Citrate buffer (pH 6.0) at 95°C for 15-20 minutes

  • Enzymatic retrieval: Proteinase K (10-20 μg/ml) for 10-15 minutes at room temperature

  • For formalin-fixed paraffin-embedded tissues, citrate buffer retrieval is typically required

• Blocking protocol modifications:

  • Use 3-5% BSA rather than serum-based blocking for membrane proteins

  • Include 0.1% Tween-20 in blocking solutions to reduce non-specific binding

  • Extended blocking (2 hours at room temperature or overnight at 4°C) improves specificity

When optimizing MBTPS2 immunostaining, researchers should test multiple antibodies as epitope accessibility can vary significantly depending on fixation method. Antibodies detecting different domains (N-terminal vs. C-terminal) may require different protocols for optimal results .

How can researchers distinguish between MBTPS2 isoforms or post-translational modifications?

Distinguishing MBTPS2 isoforms and modifications requires strategic experimental approaches:

• Isoform-specific detection strategy:

  • Use isoform-specific antibodies targeting unique regions

  • Employ reverse transcription PCR with isoform-specific primers

  • Perform immunoprecipitation followed by mass spectrometry

  • Run gradient gels (6-12%) for optimal separation of closely migrating isoforms

• Post-translational modification analysis:

  • Phosphorylation: Use phospho-specific antibodies or phosphatase treatments

  • Glycosylation: Employ PNGase F or Endoglycosidase H treatments prior to Western blot

  • Ubiquitination: Perform immunoprecipitation under denaturing conditions

  • SUMOylation: Use SUMO-specific antibodies in co-immunoprecipitation experiments

• Domain-specific antibody approach:

  • N-terminal domain antibodies (such as Aviva Systems ARP47191_P050)

  • C-terminal domain antibodies

  • Catalytic site-specific antibodies

  • Compare migration patterns and signal intensities across antibodies

• Functional validation methodologies:

  • Site-directed mutagenesis of predicted modification sites

  • Expression of truncated constructs lacking specific domains

  • Inhibitor studies targeting specific modifications (kinase inhibitors for phosphorylation)

When analyzing MBTPS2 modifications, researchers should be aware that the calculated molecular weight (57.4 kDa) may differ from observed migration patterns due to post-translational modifications. Abnormal migration patterns may provide valuable clues about protein regulation in different physiological or pathological states .

What are the best practices for quantifying MBTPS2 protein levels in various sample types?

Accurate quantification of MBTPS2 requires attention to several methodological details:

• Western blot quantification protocol:

  • Use recombinant MBTPS2 protein standards for absolute quantification

  • Include loading controls appropriate for your sample type (β-actin for general loading, calnexin for ER fractions)

  • Employ fluorescent secondary antibodies for wider linear detection range

  • Perform technical triplicates and biological replicates (minimum n=3)

• ELISA-based quantification approach:

  • Several antibodies are specifically validated for ELISA applications

  • Generate standard curves using recombinant protein

  • Optimize sample dilutions to ensure readings fall within the linear range

  • Include spike-in controls to assess matrix effects

• Mass spectrometry quantification:

  • Use stable isotope-labeled peptide standards

  • Focus on unique peptides that distinguish MBTPS2 from related proteases

  • Consider parallel reaction monitoring for targeted quantification

  • Account for varied extraction efficiency across tissue types

• Sample preparation considerations by tissue type:

  Tissue/Sample Type	Recommended Lysis Buffer	Special Considerations
  Liver	RIPA with 1% NP-40	High endogenous expression, useful as positive control
  Brain	0.5% CHAPS buffer	Requires gentle solubilization to maintain integrity
  Cell lines	NP-40 buffer (0.5-1%)	Optimization based on cell type
  FFPE tissues	Citrate buffer extraction	Extended antigen retrieval required

For accurate cross-sample comparisons, researchers should standardize sample collection procedures, as MBTPS2 levels may fluctuate with feeding/fasting cycles in metabolically active tissues and stress conditions in various cell types .

How should researchers troubleshoot weak or non-specific MBTPS2 antibody signals?

Addressing common challenges with MBTPS2 detection requires systematic troubleshooting:

• Weak signal troubleshooting protocol:

  • Increase antibody concentration incrementally (1:1000 → 1:500 → 1:250)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use signal enhancement systems (biotin-streptavidin amplification)

  • Optimize protein extraction using different detergent combinations

  • Increase protein loading (up to 50-80 μg for tissues with low expression)

• Non-specific binding resolution:

  • Test multiple blocking agents (5% BSA often superior to milk for membrane proteins)

  • Include additional wash steps (5x 10 minutes with 0.1% Tween-20)

  • Perform antibody pre-adsorption with blocking peptides when available

  • Consider more stringent antibody dilution buffer (add 150-500 mM NaCl)

• Background reduction strategies:

  • For immunohistochemistry: Implement endogenous peroxidase blocking (3% H₂O₂, 10 min)

  • For immunofluorescence: Include 0.1-0.3 M glycine to quench autofluorescence

  • Use 0.05% Tween-20 in antibody diluents

  • Consider quenching solutions for tissues with high background (liver, kidney)

• Controls for troubleshooting:

  • Positive control tissues/cells (liver extracts typically show strong MBTPS2 expression)

  • Negative controls (secondary antibody only, isotype controls)

  • Competitive blocking with immunizing peptide

  • MBTPS2 knockdown/knockout validation samples

If multiple antibodies yield inconsistent results, researchers should prioritize antibodies with literature validation and citations. The Cell Signaling Technology antibody has 25 citations, suggesting reliable performance in published research .

How can researchers validate MBTPS2 enzymatic activity in addition to protein expression?

Assessing MBTPS2 functional activity requires methodologies beyond simple protein detection:

• Substrate cleavage assay protocol:

  • Express SREBP-reporter constructs containing MBTPS2 cleavage sites

  • Monitor cleavage products by Western blot using domain-specific antibodies

  • Compare processing efficiency under sterol depletion vs. repletion conditions

  • Include S1P inhibitors as controls to ensure specificity of the MBTPS2 (S2P) cleavage step

• Cell-based activity reporter systems:

  • Implement fluorescent or luminescent reporters downstream of SREBP responsive elements

  • Compare reporter activity in wild-type vs. MBTPS2-inhibited conditions

  • Design dose-response experiments with known MBTPS2 modulators

  • Normalize to constitutive reporters to account for cell number/transfection efficiency

• In vitro proteolytic activity methodology:

  • Isolate membrane fractions containing MBTPS2

  • Incubate with fluorogenic peptide substrates containing cleavage recognition sequences

  • Monitor peptide cleavage by fluorescence/HPLC

  • Include appropriate controls (heat-inactivated enzyme, specific inhibitors)

• Correlation analysis approach:

  • Compare MBTPS2 protein levels with downstream pathway activation markers

  • Measure SREBP target genes (LDLR, HMGCR, FASN) by qPCR

  • Assess pathway activation by measuring cellular cholesterol synthesis rates

  • Determine ER stress response efficiency through ATF6 target gene induction

What are the key considerations when comparing MBTPS2 antibody performance across different research applications?

Systematic evaluation of MBTPS2 antibodies requires application-specific assessment:

• Cross-application performance matrix:

  Application	Key Performance Indicators	Recommended Antibody Types
  Western Blot	Band specificity, signal-to-noise ratio	Monoclonal for specificity
  Immunohistochemistry	Tissue distribution consistency, background	Polyclonal for signal strength
  Immunofluorescence	Subcellular localization pattern	Antibodies validated for IF (several available)
  Flow Cytometry	Population separation, signal intensity	Directly conjugated antibodies
  Immunoprecipitation	Pull-down efficiency, non-specific binding	Santa Cruz sc-293341 validated for IP

• Species cross-reactivity validation approach:

  • Test across human, mouse, and rat samples systematically

  • Compare sequence homology at epitope regions

  • Verify with species-specific positive controls

  • Consider species-specific optimization of incubation conditions

• Epitope accessibility considerations:

  • N-terminal antibodies: Better for cleaved fragments detection

  • Transmembrane region antibodies: Often problematic due to hydrophobicity

  • C-terminal antibodies: Useful for distinguishing processing states

  • Compare native vs. denatured detection efficiency

• Quantitative comparison methodology:

  • Standardize protein amounts and exposure settings

  • Implement side-by-side testing under identical conditions

  • Use recombinant standards for calibration

  • Calculate detection limits and linear range for each antibody

When selecting antibodies for new applications, researchers should prioritize those with validation data specifically for their application of interest. For example, the Proteintech antibody (12692-1-AP) is specifically validated for ELISA applications with human, mouse, and rat samples , making it an appropriate choice for these specific applications.

What emerging research directions are utilizing MBTPS2 antibodies in novel ways?

MBTPS2 antibody applications are expanding into several cutting-edge research areas:

• Single-cell analysis applications: Emerging techniques combining immunofluorescence with single-cell transcriptomics are revealing cell-type-specific MBTPS2 activation patterns in heterogeneous tissues. This approach is particularly valuable for understanding differential stress responses across specialized cell populations.

• Proximity labeling methodologies: BioID and APEX2 proximity labeling techniques coupled with MBTPS2 antibodies for verification are uncovering novel interaction partners and regulatory proteins in the MBTPS2 microenvironment, extending our understanding beyond known SREBP pathway components.

• In vivo imaging adaptations: Development of near-infrared fluorophore-conjugated MBTPS2 antibodies for in vivo imaging is enabling longitudinal studies of proteolytic activity in metabolic disease models, particularly in liver and adipose tissues where SREBP signaling is critical.

• Therapeutic targeting validation: MBTPS2 antibodies are increasingly employed to validate target engagement in therapeutic development efforts, particularly for compounds targeting disorders of cholesterol metabolism and specific dermatological conditions linked to MBTPS2 mutations.