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 .
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:
Characteristic | Specification |
---|---|
Host species | Rabbit |
Clonality | Polyclonal |
Target sequence | AA 17-70 |
Reactivity | Human |
Conjugate | FITC |
Purification | >95%, Protein G purified |
Immunogen | Recombinant Human Protein S100-P protein (17-70AA) |
Isotype | IgG |
These antibodies are highly purified (>95%) using Protein G affinity chromatography to ensure specificity and minimal background in immunodetection applications .
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 .
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 .
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 .
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 .
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 .
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:
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 .
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 .
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 .
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.
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:
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:
This research direction could significantly advance understanding of the complex relationship between ER stress pathways and colorectal cancer development, potentially identifying new therapeutic strategies .
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 .