FERMT1 Antibody, FITC conjugated

What is FERMT1 and what cellular functions does it regulate?

FERMT1 (also known as kindlin-1, KIND1, C20orf42, UNC112A, and URP1) is a member of the kindlin family of focal adhesion proteins that mediates integrin-dependent cell adhesion and signaling . FERMT1 plays pivotal roles in various cellular processes including:

• Cell adhesion and migration through integrin activation

• Regulation of cell cycle progression, particularly at the G0/G1 phase transition

• Modulation of cellular metabolism, including glycolysis and mitochondrial respiration

• Maintenance of stem cell-like properties in cancer cells

• Regulation of inflammatory responses through interaction with the NLRP3/NF-κB pathway

FERMT1 contains a FERM domain that is critical for its interaction with integrins and other binding partners. Dysfunction or dysregulation of FERMT1 has been implicated in various pathological conditions, including cancer progression and inflammatory disorders .

What are the standard applications for FERMT1 antibodies in research?

FERMT1 antibodies have been validated for multiple research applications, with specific methodological considerations for each:

• ELISA (Enzyme-Linked Immunosorbent Assay): Useful for quantitative detection of FERMT1 in cell lysates or tissue homogenates .

• Immunohistochemistry (IHC): Applied to formalin-fixed, paraffin-embedded tissue sections to analyze FERMT1 expression patterns in normal and pathological tissues .

• Immunofluorescence (IF): Enables visualization of FERMT1 subcellular localization, particularly at focal adhesions .

• Western Blotting: Used to assess FERMT1 protein expression levels and validate knockdown efficiency in experimental models .

• Immunoprecipitation: Helps identify protein-protein interactions involving FERMT1.

• Flow Cytometry: Particularly valuable for FITC-conjugated antibodies to analyze FERMT1 expression in conjunction with other cellular markers.

Each application requires specific optimization of antibody concentration, incubation time, and buffer conditions for optimal signal-to-noise ratio.

How should FERMT1 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of FERMT1 antibodies is crucial for maintaining their specificity and sensitivity:

• Storage Temperature: Store at 2°C to 8°C for frequent use. For long-term storage (up to 12 months), maintain at -20°C .

• Avoid Freeze/Thaw Cycles: Repeated freeze/thaw cycles can denature antibodies and reduce their binding efficiency .

• Preservative: FERMT1 antibodies typically contain preservatives (e.g., 0.03% Proclin) to prevent microbial contamination .

• Working Dilutions: For FITC-conjugated antibodies, optimal working dilutions should be determined experimentally for each application, but typically range from 1:50-1:500 for immunofluorescence.

• Protection from Light: FITC-conjugated antibodies are photosensitive and should be protected from light exposure during storage and handling to prevent photobleaching.

• Centrifugation: Brief centrifugation before opening is recommended to bring down any solution that might be in the cap.

What controls should be included when using FERMT1 antibodies?

Appropriate controls are essential for reliable interpretation of results:

• Positive Control: Use cell lines or tissues known to express FERMT1, such as U-251 MG or T98G glioma cells .

• Negative Control: Include samples from FERMT1 knockdown experiments (using shRNA or siRNA) .

• Isotype Control: Include an irrelevant antibody of the same isotype (IgG) and host species (rabbit) to assess non-specific binding .

• Secondary Antibody-Only Control: For indirect detection methods, include a control without primary antibody to evaluate background signal.

• Blocking Peptide Control: Pre-incubation of the antibody with its immunizing peptide should abolish specific staining.

These controls help distinguish specific signal from background or non-specific binding, ensuring reliable experimental outcomes.

What is the recommended validation approach for a new FERMT1 antibody?

When working with a new batch or source of FERMT1 antibody, validation is crucial:

• Western Blot Validation: Confirm antibody detects a band of appropriate molecular weight (~77 kDa for FERMT1) .

• RNAi Validation: Compare staining between control and FERMT1 knockdown samples (using validated siRNA or shRNA) .

• Cross-Reactivity Assessment: Test antibody specificity across multiple species if cross-reactivity is claimed.

• Peptide Competition: Pre-incubate antibody with immunizing peptide to confirm binding specificity.

• Comparative Analysis: Compare staining patterns with other validated FERMT1 antibodies.

• Application Testing: Validate performance in each intended application (ELISA, IHC, IF, etc.) .

Documentation of validation experiments should be maintained according to research reproducibility standards.

What methods are most effective for FERMT1 knockdown validation in experimental models?

Based on published research, effective FERMT1 knockdown can be achieved and validated through:

• siRNA-Mediated Knockdown:

  • FERMT1-specific siRNA sequence (CAGAAGAACUUUCAUUGUUTT-AACAAUGAAAGUUCUUCUGTT) has demonstrated effective knockdown in rat models and cell lines .

  • Transfection with lipid-based reagents should be optimized for each cell type.

  • For in vivo applications, direct injection into target tissues (e.g., 500 pmol/10 μl into brain ventricles) has proven effective .

• shRNA-Mediated Stable Knockdown:

  • Lentiviral delivery of FERMT1-targeting shRNA provides sustained knockdown for long-term experiments .

  • Selection of the most effective shRNA sequence through preliminary screening is recommended .

• Validation Methods:

  • qRT-PCR to assess mRNA reduction (typically 70-90% reduction is achievable) .

  • Western blot to confirm protein reduction, using rabbit polyclonal antibodies that recognize the full protein or specific domains .

  • Immunofluorescence to visualize reduced protein expression at the cellular level .

Table 1: Comparison of FERMT1 Knockdown Methods

How can FERMT1 antibodies be used to investigate cancer stem cell properties?

FERMT1 has been implicated in regulating cancer stem cell (CSC) properties, particularly in glioma. FITC-conjugated FERMT1 antibodies can facilitate several experimental approaches:

• Sphere Formation Assays:

  • FERMT1 knockdown significantly reduces sphere diameter in glioma cell lines, suggesting impaired CSC self-renewal .

  • FERMT1 antibodies can be used to monitor protein expression throughout sphere formation.

  • Quantitative analysis of sphere diameter should be performed using standardized imaging techniques.

• CSC Marker Co-expression Analysis:

  • Flow cytometry with FITC-conjugated FERMT1 antibodies can be combined with other fluorophore-conjugated CSC markers (e.g., CD44) .

  • FERMT1 knockdown reduces CD44 expression in glioma cells, suggesting its role in CSC maintenance .

• Pluripotency Factor Expression:

  • FERMT1 regulates expression of MYC, OCT4, and NANOG in glioma cells .

  • Immunofluorescence co-staining with FITC-conjugated FERMT1 and antibodies against these transcription factors can reveal correlations at single-cell level.

• Metabolic Profiling:

  • FERMT1 influences cellular metabolism in glioma, affecting glycolysis and mitochondrial respiration .

  • Combining FITC-conjugated FERMT1 staining with metabolic probes can reveal relationships between FERMT1 expression and metabolic phenotypes.

Table 2: Effect of FERMT1 Knockdown on Stem Cell Properties in Glioma

What are the optimal protocols for detecting FERMT1 using FITC-conjugated antibodies in immunofluorescence applications?

For optimal detection of FERMT1 using FITC-conjugated antibodies in immunofluorescence:

• Cell Fixation:

  • 4% paraformaldehyde (PFA) for 15-20 minutes at room temperature preserves FERMT1 structure and localization.

  • For membrane-associated FERMT1, milder fixation with 2% PFA may improve epitope accessibility.

• Permeabilization:

  • 0.1-0.3% Triton X-100 for 10 minutes for intracellular access.

  • For focal adhesion-associated FERMT1, gentler permeabilization with 0.1% saponin may better preserve structural integrity.

• Blocking:

  • 5-10% normal serum (from species unrelated to primary and secondary antibodies) with 1% BSA for 1 hour.

  • Include 0.1% Tween-20 to reduce non-specific binding.

• Antibody Dilution and Incubation:

  • Optimal dilution of FITC-conjugated FERMT1 antibodies typically ranges from 1:50 to 1:200.

  • Incubate overnight at 4°C in a humidified chamber protected from light.

• Counterstaining:

  • DAPI (1 μg/ml) for nuclear visualization.

  • Phalloidin-conjugated dyes (different fluorophore) for actin cytoskeleton visualization, which often colocalizes with FERMT1 at focal adhesions.

• Mounting:

  • Anti-fade mounting medium to prevent photobleaching of FITC.

  • Consider ProLong Gold or similar media containing anti-fade reagents.

• Image Acquisition:

  • Use appropriate excitation (488 nm) and emission (515-530 nm) filters for FITC.

  • Acquire Z-stacks for accurate localization, particularly for focal adhesion studies.

How can FERMT1 antibodies be used to investigate inflammatory pathways?

FERMT1 has been implicated in inflammatory processes, particularly through the NLRP3/NF-κB pathway. FITC-conjugated FERMT1 antibodies can be employed to study these relationships:

• Co-localization Studies:

  • Dual immunofluorescence with FITC-conjugated FERMT1 antibodies and antibodies against inflammatory markers (NLRP3, ASC, cleaved-caspase-1) .

  • Confocal microscopy can reveal spatial relationships between FERMT1 and inflammasome components.

• Cell-Specific Expression Analysis:

  • In microglia, FERMT1 knockdown reduces activation markers (e.g., Iba1) and inflammatory cytokine production (IL-1β, IL-18) .

  • Flow cytometry with FITC-conjugated FERMT1 antibodies can identify FERMT1-expressing cell populations in heterogeneous samples.

• Activation State Assessment:

  • FERMT1 knockdown prevents NF-κB pathway activation by inhibiting phosphorylation of IκBa and NF-κB p65 .

  • Combining FITC-conjugated FERMT1 staining with phospho-specific antibodies can reveal correlations between FERMT1 expression and NF-κB activation at single-cell level.

• Intervention Studies:

  • FITC-conjugated FERMT1 antibodies can be used to track changes in protein expression following treatment with anti-inflammatory compounds.

  • Time-course experiments can reveal dynamic changes in FERMT1 expression during inflammatory responses.

Table 3: Effect of FERMT1 Knockdown on Inflammatory Markers

What methods can be used to quantify FERMT1 expression changes using FITC-conjugated antibodies?

Quantitative analysis of FERMT1 expression using FITC-conjugated antibodies requires rigorous methodological approaches:

• Flow Cytometry Quantification:

  • Mean Fluorescence Intensity (MFI) provides a population-level measure of FERMT1 expression.

  • Standardize measurements using calibration beads with known fluorophore amounts.

  • Include isotype control to determine background fluorescence levels.

  • Calculate relative expression as ratio of sample MFI to control MFI.

• Image-Based Quantification:

  • Confocal or fluorescence microscopy with consistent acquisition parameters.

  • Measure integrated density (product of area and mean gray value) of FERMT1-positive regions.

  • Normalize to cell number (e.g., DAPI-positive nuclei) or cell area.

  • Use automated image analysis software (ImageJ, CellProfiler) with standardized thresholding algorithms.

• Homogeneous Time-Resolved Fluorescence (HTRF):

  • Can be adapted for FERMT1 quantification similar to the method described for actin binding proteins .

  • Combines time-resolved measurement with FRET between a donor (e.g., Terbium cryptate) and FITC as acceptor.

  • Allows high-throughput quantification in plate-based format.

• Tissue Microarray Analysis:

  • Simultaneous analysis of multiple tissue samples under identical staining conditions.

  • Scoring systems can be developed based on intensity and distribution of FERMT1 staining.

  • Digital pathology approaches enable automated quantification across large sample sets.

Table 4: Quantification Methods for FERMT1-FITC Antibody Signal

How can FERMT1 antibodies be incorporated into multiplex immunofluorescence panels?

Incorporating FITC-conjugated FERMT1 antibodies into multiplex panels requires careful planning:

• Spectral Compatibility:

  • FITC emission (peak ~520 nm) should be sufficiently separated from other fluorophores.

  • Compatible fluorophores include DAPI (~460 nm), Cy3 (~570 nm), Cy5 (~670 nm), and APC (~660 nm).

  • Consider spectral unmixing for fluorophores with overlapping emission spectra.

• Panel Design:

  • For studying FERMT1 in cancer stem cells, combine with antibodies against CD44, MYC, OCT4, or NANOG .

  • For inflammatory pathways, combine with antibodies against NLRP3, ASC, cleaved-caspase-1, or NF-κB pathway components .

  • For focal adhesion studies, combine with antibodies against integrins, talin, or paxillin.

• Sequential Staining Protocol:

  • Apply antibodies sequentially for markers requiring different fixation or retrieval conditions.

  • Use dedicated blocking steps between antibody applications to prevent cross-reactivity.

  • Consider tyramide signal amplification (TSA) for low-abundance targets.

• Controls for Multiplex Panels:

  • Single-color controls to establish appropriate compensation settings.

  • Fluorescence minus one (FMO) controls to set accurate gating boundaries.

  • Absorption controls to detect and correct for potential energy transfer between fluorophores.

• Image Acquisition and Analysis:

  • Use multispectral imaging systems (e.g., Vectra, Mantra) for optimal separation of fluorophores.

  • Employ automated cell segmentation and quantification algorithms.

  • Analyze co-localization using Pearson's correlation coefficient or Mander's overlap coefficient.

What are the experimental considerations when using FERMT1 antibodies to study cellular metabolism?

FERMT1 has been implicated in regulating cellular metabolism, particularly glycolysis and mitochondrial respiration . FITC-conjugated FERMT1 antibodies can be employed to investigate these relationships:

• Co-staining with Metabolic Markers:

  • Combine FITC-conjugated FERMT1 antibodies with antibodies against glycolysis-related proteins (GLUT1, GLUT3, GLUT4, SCO2) .

  • Use mitochondrial dyes (e.g., MitoTracker) to assess relationships between FERMT1 expression and mitochondrial content or membrane potential.

• Functional Metabolic Assays:

  • Sort cells based on FERMT1-FITC staining intensity for downstream metabolic analysis.

  • Measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in sorted populations.

  • Analyze ATP production, glucose uptake, lactate production, and G6PDH activity in relation to FERMT1 expression levels .

• Live-Cell Imaging:

  • For dynamic studies, consider using cell-permeable FERMT1 antibody fragments conjugated to FITC.

  • Combine with fluorescent metabolic sensors (e.g., FRET-based ATP sensors) for real-time correlation studies.

• Metabolism-Related Experimental Conditions:

  • Control for cell confluence, as contact inhibition affects both FERMT1 localization and metabolic profiles.

  • Standardize glucose concentration in media, as FERMT1's effects on glycolysis may be glucose-dependent.

  • Account for oxygen levels, as hypoxia alters both FERMT1 expression and metabolic pathways.

Table 5: Metabolic Parameters Affected by FERMT1 Knockdown