MBTPS1 Antibody, FITC conjugated

What is MBTPS1 and why is it relevant to cancer research?

MBTPS1 (Membrane-Bound Transcription Factor Peptidase, Site 1), also known as SKI-1/S1P, is a calcium-dependent serine protease that plays a critical role in regulating cellular functions. In colorectal cancer, MBTPS1 has been demonstrated to regulate cancer cell proliferation primarily through SREBP-associated lipid metabolism pathways . The protein has significant research interest because knockout studies reveal severe attenuation of proliferation and marked downregulation of energy metabolism pathways in colon cancer cells . MBTPS1 is widely expressed across many tissue types and functions by cleaving after hydrophobic or small residues when Arg or Lys is in position P4 .

The canonical MBTPS1 protein in humans consists of 1052 amino acid residues with a molecular mass of approximately 117.7 kDa . Its subcellular localization is primarily in the endoplasmic reticulum (ER) and Golgi apparatus, where it processes various substrates involved in lipid metabolism, protein processing, and cellular stress responses .

What are the specifications of commercially available FITC-conjugated MBTPS1 antibodies?

FITC-conjugated MBTPS1 antibodies, such as the rabbit polyclonal antibody targeting amino acids 17-70 (ABIN7364854), are designed for immunodetection of human MBTPS1 . These antibodies typically have the following specifications:

These antibodies are highly purified (>95%) using Protein G affinity chromatography to ensure specificity and minimal background in immunodetection applications .

What experimental applications are suitable for FITC-conjugated MBTPS1 antibodies?

FITC-conjugated MBTPS1 antibodies are versatile tools for various research applications focusing on the detection and localization of MBTPS1 protein. Suitable experimental applications include:

• Immunofluorescence (IF): Direct visualization of MBTPS1 in fixed cells without need for secondary antibodies, ideal for localization studies in the ER and Golgi .

• Flow Cytometry (FCM): Detection of MBTPS1 in cell populations, enabling quantitative analysis of expression levels across different cell types or experimental conditions.

• Immunohistochemistry (IHC): Examination of MBTPS1 expression in tissue sections, particularly valuable for cancer research .

• Confocal Microscopy: High-resolution imaging of MBTPS1 subcellular localization, especially useful for co-localization studies with other ER/Golgi markers.

The direct FITC conjugation eliminates the need for secondary antibody incubation steps, reducing experimental time and potential cross-reactivity issues in multi-labeling experiments .

What are the optimal conditions for using FITC-conjugated MBTPS1 antibodies in immunofluorescence?

When using FITC-conjugated MBTPS1 antibodies for immunofluorescence, researchers should consider the following methodological approach:

• Fixation: Use 4% paraformaldehyde for 15-20 minutes at room temperature to preserve cellular architecture while maintaining epitope accessibility.

• Permeabilization: Apply 0.1-0.5% Triton X-100 for 5-10 minutes to allow antibody access to intracellular compartments where MBTPS1 resides (ER and Golgi).

• Blocking: Incubate with 5-10% normal serum (from a species different from the antibody host) with 1% BSA for 30-60 minutes to minimize non-specific binding.

• Antibody Dilution: Typically 1:50 to 1:200 dilution in blocking buffer, though optimal concentration should be determined empirically for each application.

• Incubation Time: Overnight at 4°C or 1-2 hours at room temperature in a humidified chamber protected from light to prevent photobleaching of the FITC fluorophore.

• Counterstaining: Use DAPI (1 μg/ml) for nuclear visualization and phalloidin-conjugated dyes for F-actin when cell morphology assessment is required.

• Mounting: Use anti-fade mounting medium to preserve fluorescence signal intensity over time.

For optimal imaging, use filter sets appropriate for FITC (excitation ~495 nm, emission ~519 nm) and avoid prolonged exposure to excitation light to prevent photobleaching .

How can researchers validate the specificity of MBTPS1 antibodies in their experimental models?

Validation of MBTPS1 antibody specificity is crucial for research reliability. A comprehensive validation approach should include:

• Positive Controls: Use cell lines known to express MBTPS1 at detectable levels, such as HT-29 colorectal cancer cells mentioned in the literature .

• Negative Controls:

  • Omit primary antibody while maintaining all other steps

  • Use isotype control antibodies (rabbit IgG-FITC) at the same concentration

  • Include MBTPS1 knockout or knockdown samples where available

• Western Blot Verification: Confirm antibody recognizes a single band of appropriate molecular weight (~117.7 kDa) . This cross-validation with a different technique strengthens confidence in antibody specificity.

• Peptide Competition Assay: Pre-incubate antibody with excess immunizing peptide before application to samples; specific signal should be significantly reduced.

• siRNA Knockdown: Confirm reduced signal intensity correlates with MBTPS1 knockdown efficiency measured by other methods (qPCR, Western blot).

• Multi-antibody Concordance: Compare staining patterns using antibodies targeting different epitopes of MBTPS1 (e.g., AA 17-70 vs. AA 803-1052) .

Thorough validation ensures that observed signals genuinely represent MBTPS1 distribution and not artifacts or cross-reactivity with other proteins .

What are the considerations for using FITC-conjugated MBTPS1 antibodies in multicolor flow cytometry?

When incorporating FITC-conjugated MBTPS1 antibodies into multicolor flow cytometry panels, researchers should address these methodological considerations:

• Spectral Overlap: FITC (emission peak ~519 nm) has potential overlap with PE (emission peak ~575 nm) and other green-yellow fluorophores. Implement proper compensation controls using single-stained samples.

• Panel Design:

  • Position FITC in a channel detecting abundant proteins (like MBTPS1 in cancer cells) rather than rare markers

  • Avoid pairing with tandem dyes that use FITC as a donor (e.g., PE-Cy5)

  • Consider brightness hierarchy when designing panels (FITC has moderate brightness)

• Fixation/Permeabilization: Since MBTPS1 is primarily intracellular (ER/Golgi), use appropriate permeabilization reagents compatible with flow cytometry:

  • Commercial kits (e.g., Cytofix/Cytoperm™)

  • Methanol/acetone for nuclear/organelle proteins

  • Saponin-based reagents for reversible permeabilization

• Titration: Perform antibody titration to determine optimal concentration that maximizes signal-to-noise ratio while minimizing reagent usage.

• Fluorescence Minus One (FMO) Controls: Essential for setting accurate gates, especially for continuous expression markers like MBTPS1.

• Viability Dye: Include far-red viability dye to exclude dead cells that may bind antibodies non-specifically.

• Autofluorescence Management: Account for increased autofluorescence in the FITC channel, particularly in larger or more granular cells. Consider using unstained controls matched to each cell type being analyzed .

How does MBTPS1 regulate lipid metabolism in colorectal cancer cells?

MBTPS1 regulates lipid metabolism in colorectal cancer cells primarily through processing SREBP transcription factors, which are master regulators of lipogenic pathways. Research demonstrates several key mechanistic aspects:

• SREBP Activation: MBTPS1 proteolytically processes SREBP precursors (SREBP1 and SREBP2) in coordination with S2P, releasing the active transcription factor domains that translocate to the nucleus .

• Lipogenic Gene Expression: Activated SREBPs upregulate genes involved in fatty acid synthesis, including FASN, ACC, and SCD1, which are frequently overexpressed in colorectal cancer .

• Cancer Cell Proliferation: Knockdown of SREBPs in colorectal cancer cells significantly reduces fatty acid synthesis rates, impairs cell proliferation, and inhibits the ability to form spheroids – an in vitro measure of cancer stemness .

• Xenograft Growth Inhibition: SREBP pathway inhibition suppresses growth and lipogenesis in colon cancer xenograft models, indicating essential roles in in vivo tumor development .

• Metabolic Reprogramming: MBTPS1 knockout in HT-29 colon cancer cells causes severe attenuation of proliferation with marked downregulation of energy metabolism pathways, suggesting MBTPS1-processed SREBPs are critical for maintaining cancer metabolic programs .

These findings indicate that MBTPS1 controls colorectal cancer growth by facilitating SREBP-mediated lipid synthesis, which provides crucial membrane components and signaling molecules necessary for rapid cell division .

What is the connection between MBTPS1, interferon pathways, and cancer cell survival?

Research has revealed an unexpected connection between MBTPS1, interferon pathways, and cancer cell survival that presents interesting therapeutic implications:

• MBTPS1 Knockout Phenotype: Complete MBTPS1 gene knockout in colon cancer cells resulted in severely attenuated proliferation, with only a single clone surviving the knockout procedure .

• Interferon Pathway Upregulation: The surviving MBTPS1 knockout clone exhibited significant upregulation of the type-1 interferon pathway, suggesting an adaptive response .

• Survival Dependency: Experimental inhibition of the upregulated type-1 interferon pathway in MBTPS1 knockout cells completely halted proliferation, indicating this pathway became essential for survival in the absence of MBTPS1 .

• Partial Rescue Effects: When MBTPS1 expression was partially restored in knockout cells, researchers observed:

  • Proportional recovery of proliferation capacity

  • Corresponding increase in SREBP levels

  • Partial normalization of metabolic pathways

This compensatory relationship suggests that cancer cells can activate interferon signaling as an alternative survival pathway when MBTPS1-dependent lipid metabolism is compromised. This finding has significant implications for combination therapy approaches targeting both MBTPS1 and interferon signaling pathways simultaneously .

How can FITC-conjugated MBTPS1 antibodies be used to study MBTPS1 trafficking and processing?

FITC-conjugated MBTPS1 antibodies offer powerful tools for investigating MBTPS1 trafficking and processing dynamics through these methodological approaches:

• Live-Cell Imaging: Using cell-permeable FITC-conjugated antibodies to monitor MBTPS1 movement between cellular compartments in real-time:

  • Tracking ER-to-Golgi transport under normal conditions

  • Observing responses to cellular stressors or drug treatments

  • Measuring kinetics of MBTPS1 redistribution

• Colocalization Studies: Combining FITC-conjugated MBTPS1 antibodies with markers for different subcellular compartments using confocal microscopy:

  • ER markers (e.g., calnexin, PDI)

  • Golgi markers (e.g., GM130, TGN46)

  • SREBP substrates to monitor enzyme-substrate interactions

• FRET/FLIM Analysis: When used with acceptor fluorophore-labeled substrate proteins, FITC-conjugated MBTPS1 antibodies can enable Förster Resonance Energy Transfer (FRET) or Fluorescence-Lifetime Imaging Microscopy (FLIM) to detect direct molecular interactions.

• Pulse-Chase Experiments: Tracking newly synthesized MBTPS1 through the secretory pathway using time-course fixation and immunofluorescence.

• Drug Perturbation Studies: Examining how MBTPS1 localization and activity change in response to:

  • ER stress inducers (tunicamycin, thapsigargin)

  • Golgi transport inhibitors (brefeldin A, monensin)

  • Protease inhibitors specific to serine proteases

These techniques allow researchers to elucidate the spatial and temporal dynamics of MBTPS1, contributing to understanding its regulation and identifying potential points for therapeutic intervention .

What are the implications of MBTPS1 as a therapeutic target in colorectal cancer?

Evidence from research suggests MBTPS1 represents a promising therapeutic target in colorectal cancer with several notable implications:

• Essential Role in Cancer Metabolism: MBTPS1 knockout severely attenuates proliferation of colorectal cancer cells through disruption of SREBP-mediated lipid metabolism, indicating its fundamental role in cancer cell survival .

• Limited Redundancy: The observation that only a single clone survived MBTPS1 knockout suggests limited redundancy in its function, potentially reducing bypass mechanisms that often develop in targeted therapies .

• Synergistic Opportunities: The compensatory upregulation of type-1 interferon pathway in MBTPS1-deficient cells presents opportunities for combination therapy approaches targeting both pathways .

• Broader Cancer Relevance: Given that SREBP-dependent lipid metabolism is elevated in many cancer types, MBTPS1 inhibition could have applications beyond colorectal cancer.

• Potential Therapeutic Approaches:

  • Small molecule inhibitors of MBTPS1 protease activity

  • Antisense oligonucleotides targeting MBTPS1 mRNA

  • Antibody-drug conjugates using MBTPS1 antibodies as targeting moieties

• Biomarker Applications: MBTPS1 expression levels or activation status could serve as biomarkers for:

  • Patient stratification for targeted therapies

  • Monitoring treatment response

  • Predicting disease progression

The critical dependence of colorectal cancer cells on MBTPS1 for lipid metabolism and proliferation suggests that therapeutic strategies targeting this protease could provide effective new approaches for colorectal cancer treatment .

What are common issues with FITC-conjugated antibodies and how can they be addressed?

When working with FITC-conjugated MBTPS1 antibodies, researchers may encounter several technical challenges that can be methodically addressed:

• Photobleaching: FITC is relatively susceptible to photobleaching compared to other fluorophores.

  • Solution: Use anti-fade mounting media with DABCO or propyl gallate

  • Solution: Minimize exposure time during imaging

  • Solution: Consider using newer generation FITC derivatives with improved photostability

• Autofluorescence: Cellular components like NADH, flavins, and lipofuscin can generate green autofluorescence that overlaps with FITC.

  • Solution: Use Sudan Black B (0.1-0.3%) to quench lipofuscin autofluorescence

  • Solution: Apply spectral unmixing during confocal microscopy

  • Solution: Consider background subtraction using unstained controls

• pH Sensitivity: FITC fluorescence decreases at lower pH (e.g., in acidic organelles).

  • Solution: Use pH-stabilized mounting media (pH 8.0-9.0)

  • Solution: Consider pH-insensitive alternatives like Alexa Fluor 488 for acidic compartments

• Fixation-Related Issues: Certain fixatives can alter epitope structure or create aldehyde-induced autofluorescence.

  • Solution: Compare multiple fixation methods (PFA, methanol, acetone)

  • Solution: Treat with sodium borohydride (1 mg/ml) to reduce fixative-induced fluorescence

  • Solution: Optimize fixation time to balance structure preservation and antibody accessibility

• Signal Intensity: FITC has moderate brightness compared to newer fluorophores.

  • Solution: Adjust antibody concentration (perform titration experiments)

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

  • Solution: Consider signal amplification methods compatible with direct conjugates

• Cross-Reactivity: Polyclonal antibodies may exhibit some non-specific binding.

  • Solution: Increase blocking time and concentration (5-10% normal serum with 1-3% BSA)

  • Solution: Pre-adsorb antibody with tissue powder from non-relevant species

  • Solution: Include appropriate negative controls in all experiments

How can researchers optimize immunofluorescence protocols for detecting MBTPS1 in different subcellular compartments?

Detecting MBTPS1 across its different subcellular locations (primarily ER and Golgi) requires optimization of immunofluorescence protocols:

• Organelle-Specific Fixation Methods:

  • ER preservation: 4% PFA with 0.1% glutaraldehyde maintains ER architecture

  • Golgi preservation: 100% ice-cold methanol for 5 minutes preserves Golgi structure while extracting cytosolic proteins

• Permeabilization Optimization:

  • Digitonin (25-50 μg/ml): Selectively permeabilizes plasma membrane while leaving ER/Golgi intact, useful for distinguishing cytosolic vs. organelle-associated pools

  • Saponin (0.1-0.3%): Reversible, gentler permeabilization that maintains membrane integrity

  • Triton X-100 (0.1-0.5%): Stronger permeabilization for accessing less accessible epitopes

• Epitope Retrieval for Improved Detection:

  • Heat-mediated antigen retrieval (citrate buffer pH 6.0)

  • Enzymatic retrieval with proteases for heavily fixed tissues

  • SDS treatment (0.5% for 5 minutes) for unmasking certain epitopes

• Co-localization Strategy:

  • Pair FITC-MBTPS1 antibody with organelle markers in contrasting colors:

    • ER: Anti-calnexin (red fluorophore)

    • Golgi: Anti-GM130 (far-red fluorophore)

    • ER-Golgi interface: Anti-ERGIC-53 (red fluorophore)

• Confocal Acquisition Parameters:

  • Use narrow bandpass filters to minimize spectral overlap

  • Employ sequential scanning rather than simultaneous acquisition

  • Apply Nyquist sampling criteria for optimal resolution

  • Utilize Z-stacks with deconvolution for 3D localization analysis

• Signal Enhancement Methods:

  • Tyramide signal amplification compatible with FITC

  • Enhanced exposure times balanced against photobleaching risk

  • Optimal pinhole settings (0.8-1.2 Airy units) for best signal-to-noise ratio

How might MBTPS1 antibodies contribute to understanding the ATF6 pathway in colorectal cancer development?

FITC-conjugated MBTPS1 antibodies offer valuable tools for investigating the ATF6 pathway's role in colorectal cancer, with several promising research directions:

• ATF6 Processing Studies: ATF6 is a direct substrate of MBTPS1 and has been implicated in colorectal cancer development . FITC-MBTPS1 antibodies could help visualize:

  • Co-localization of MBTPS1 and ATF6 during ER stress responses

  • Temporal dynamics of their interaction during cancer progression

  • Altered processing in response to therapeutic interventions

• Mechanistic Investigations: Research indicates that mice with intestinal epithelial expression of active ATF6 develop spontaneous colon adenomas by 12 weeks of age . MBTPS1 antibodies could help elucidate:

  • Whether MBTPS1 expression/activity correlates with ATF6 activation in tumor development

  • Spatial distribution of MBTPS1 in pre-neoplastic lesions versus established tumors

  • Changes in MBTPS1-ATF6 interaction during adenoma-to-carcinoma progression

• Prognostic Marker Development: Increased ATF6 expression is associated with reduced disease-free survival in colorectal cancer patients . Researchers could use MBTPS1 antibodies to:

  • Develop dual-staining protocols for patient tissue microarrays

  • Correlate MBTPS1/ATF6 co-expression patterns with clinical outcomes

  • Create multiparameter prognostic tools combining MBTPS1, ATF6, and established markers

• Therapeutic Vulnerability Identification: MBTPS1 antibodies could help identify patient subgroups that might benefit from targeted therapies by:

  • Characterizing how the MBTPS1-ATF6 axis intersects with the oncogenic CIP2A pathway known to increase cancer cell survival

  • Determining whether MBTPS1 inhibition synergizes with ATF6 pathway modulation

  • Identifying biomarkers of response to ER stress-targeting therapies

This research direction could significantly advance understanding of the complex relationship between ER stress pathways and colorectal cancer development, potentially identifying new therapeutic strategies .

What emerging methods might enhance the utility of FITC-conjugated MBTPS1 antibodies in cancer research?

Several cutting-edge methodologies could substantially expand the research applications of FITC-conjugated MBTPS1 antibodies:

• Super-Resolution Microscopy Techniques:

  • STORM (Stochastic Optical Reconstruction Microscopy): Can achieve 20-30 nm resolution to precisely map MBTPS1 distribution within ER/Golgi subdomains

  • Expansion Microscopy: Physical tissue expansion allows standard confocal microscopes to achieve super-resolution imaging of MBTPS1 subcellular localization

  • STED (Stimulated Emission Depletion): Compatible with FITC fluorophores for nanoscale imaging of MBTPS1 organization

• Intravital Imaging Applications:

  • Real-time visualization of MBTPS1 dynamics in tumor xenograft models

  • Tracking MBTPS1 activity in response to drug treatments in living organisms

  • Monitoring changes in MBTPS1 expression during tumor progression and metastasis

• Multiplex Tissue Analysis:

  • Cyclic Immunofluorescence: Sequential staining/imaging/bleaching cycles to analyze dozens of markers alongside MBTPS1

  • CODEX (CO-Detection by indEXing): DNA-barcoded antibody technology for highly multiplexed tissue imaging including MBTPS1

  • Imaging Mass Cytometry: Combining FITC-MBTPS1 antibodies with metal-tagged antibodies for highly multiplexed analysis

• Single-Cell Analytical Methods:

  • Integration with single-cell RNA-seq to correlate MBTPS1 protein levels with transcriptional profiles

  • Flow cytometry sorting of cells based on MBTPS1-FITC signal followed by downstream genomic/proteomic analysis

  • Correlation of MBTPS1 levels with functional metabolic parameters at single-cell resolution

• Proximity Labeling Applications:

  • APEX2 or BioID fusion with MBTPS1 to identify proximal proteins in living cells

  • FITC-conjugated antibodies to validate proximity labeling results

  • Identification of novel MBTPS1 interaction partners in cancer-specific contexts

These emerging methods would significantly enhance spatial, temporal, and functional analyses of MBTPS1 in cancer research contexts, potentially leading to new insights into its role in disease progression and therapeutic targeting .