FMN1 Antibody, FITC conjugated

What is FMN1 protein and why is it of interest in research?

FMN1 (Formin 1) is a protein involved in cytoskeletal reorganization, particularly influencing actin filament assembly. It plays a crucial role in normal limb development, and deficiency mutations at the mammalian limb deformity (ld) locus lead to profound developmental defects in limb and kidney formation. Formins can translocate between the cytosol and nucleus in an HGF-dependent manner and are involved in establishing Sonic hedgehog/FGF-4 feedback loops in limb buds. The human Formin 1 gene maps to chromosome 15q13.3, making it relevant for developmental biology, cytoskeletal studies, and signal transduction research .

What are the common applications of FITC-conjugated FMN1 antibodies?

FITC-conjugated FMN1 antibodies are primarily used in fluorescence-based applications including:

• Immunofluorescence with paraffin-embedded tissues (IF/IHC-P)

• Immunofluorescence with frozen tissues (IF/IHC-F)

• Immunocytochemistry (ICC)

• Flow cytometry

• Fluorescence microscopy for subcellular localization studies

These applications allow direct visualization of FMN1 protein without the need for secondary antibody incubation, simplifying experimental protocols and reducing background signal .

What species reactivity can I expect from commercially available FMN1-FITC antibodies?

The reactivity profile varies between different FMN1-FITC antibody products:

When selecting an antibody, consider your experimental organism and verify cross-reactivity through literature or preliminary testing if working with species not listed as confirmed reactive .

How should I optimize staining protocols for FMN1-FITC antibodies in fluorescence microscopy?

For optimal results with FMN1-FITC antibodies in fluorescence microscopy:

• Sample preparation: Fix cells/tissues appropriately (4% paraformaldehyde for 15-20 minutes for cells; formalin fixation for tissues followed by proper antigen retrieval)

• Blocking: Use 5% BSA in PBS for 1-2 hours at room temperature to minimize non-specific binding

• Antibody dilution: Start with manufacturer-recommended dilutions (typically 1:50-1:200 for IF applications)

• Incubation conditions: Incubate overnight at 4°C in a humidified chamber protected from light to preserve FITC fluorescence

• Controls: Include negative controls (omitting primary antibody) and positive controls (tissue/cells known to express FMN1)

• Counterstaining: Use DAPI for nuclear visualization, avoiding fluorophores with spectral overlap with FITC

• Mounting: Use anti-fade mounting medium to prevent photobleaching during imaging and storage

Optimize antibody concentration through titration experiments, as excessive antibody can increase background while insufficient antibody can produce weak signals .

What are the recommended storage conditions for maintaining FITC-conjugated antibody activity?

To maintain optimal activity of FITC-conjugated FMN1 antibodies:

• Store at -20°C in the dark (FITC is light-sensitive)

• Aliquot into multiple vials upon receipt to avoid repeated freeze-thaw cycles

• Use storage buffers containing glycerol (typically 50%) to prevent freeze damage

• Some products are stored in buffers containing preservatives like 0.03% Proclin300

• When handling, keep on ice and protected from light

• For short-term storage (1-2 weeks), refrigeration at 4°C is acceptable

• Never store diluted antibody solutions for extended periods

Following these guidelines will help maintain fluorescence intensity and binding specificity throughout your research project .

How can I use FMN1-FITC antibodies to study FMN1-FNBP4 interactions in cellular contexts?

To investigate FMN1-FNBP4 interactions using FMN1-FITC antibodies:

• Co-localization studies: Combine FMN1-FITC antibody with differently-labeled FNBP4 antibody (e.g., TRITC-conjugated) for dual-color immunofluorescence microscopy

• FRET analysis: If using appropriate fluorophore pairs, analyze Förster resonance energy transfer to detect protein proximity

• Co-immunoprecipitation validation: Use unlabeled antibodies for co-IP followed by blotting with fluorescently labeled antibodies

• Domain mapping: Use antibodies targeting different domains (FH1, FH2) to determine interaction specificity

• Stimulation experiments: Assess co-localization following cytoskeletal reorganization triggers

Recent research has elucidated the binding kinetics between FNBP4 and FMN1 using SPR and ELISA. FMN1 antibodies raised against 6xHis-tagged FH1-FH2 domains were shown to detect FNBP4-FMN1 complexes in ELISA assays, suggesting applications for studying these interactions in cellular contexts .

What considerations are important when designing multiplexed immunofluorescence experiments that include FMN1-FITC antibodies?

For successful multiplexed immunofluorescence including FMN1-FITC antibodies:

• Spectral compatibility: FITC emits in the green spectrum (~520 nm), so pair with fluorophores having minimal spectral overlap (e.g., TRITC, Cy5)

• Antibody origin: Ensure antibodies for multiple targets originate from different host species to avoid cross-reactivity

• Sequential staining: Consider sequential rather than simultaneous staining if antibodies have similar hosts

• Signal intensity balancing: Adjust exposure times or antibody concentrations to balance signal intensities between channels

• Bleed-through controls: Include single-stained controls to assess and correct spectral bleed-through

• Blocking optimization: When using multiple antibodies, optimize blocking conditions to minimize background across all channels

• Order of application: Apply the most robust antibody first, followed by more sensitive ones

Careful experimental design and appropriate controls are essential for accurate interpretation of co-localization or expression pattern data .

What are common issues with FMN1-FITC antibodies and how can they be addressed?

When working with FMN1-FITC antibodies, researchers may encounter several common issues:

For definitive validation, consider comparing results with alternative FMN1 antibodies or complementary techniques such as RNA in situ hybridization .

How can I determine if my FMN1-FITC antibody is detecting the correct protein domains in experimental samples?

To validate domain-specific detection by FMN1-FITC antibodies:

• Epitope mapping: Verify which domain the antibody targets (FH1, FH2, or other domains) based on immunogen information

• Blocking peptide experiments: Pre-incubate antibody with excess immunizing peptide to confirm specificity

• Knockout/knockdown controls: Test antibody in FMN1-depleted samples (CRISPR knockout or siRNA knockdown)

• Domain deletion constructs: Express FMN1 constructs lacking specific domains to confirm binding specificity

• Western blot correlation: Perform parallel Western blot analysis to confirm molecular weight

• Alternative antibodies: Compare staining patterns with antibodies targeting different FMN1 epitopes

Available FMN1-FITC antibodies target different regions of the protein. For example, catalog #bs-13185R-FITC targets amino acids 651-750, while ABIN7153112 targets amino acids 354-487. Understanding these differences is crucial for interpretation of domain-specific functions .

What protocols can be used to study FMN1 interactions with the actin cytoskeleton using FITC-conjugated antibodies?

To investigate FMN1 interactions with the actin cytoskeleton:

• Co-staining protocol:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 for 10 minutes

  • Block with 5% BSA for 1 hour

  • Incubate with FMN1-FITC antibody (1:100 dilution) for 2 hours at room temperature

  • Co-stain with rhodamine-phalloidin (1:200) for 30 minutes to visualize F-actin

  • Counterstain nuclei with DAPI

  • Image using confocal microscopy for co-localization analysis

• Live-cell imaging approaches:

  • Transfect cells with FMN1-GFP constructs

  • Use SiR-actin for live F-actin visualization

  • Perform time-lapse imaging during cytoskeletal reorganization events

• Biochemical fractionation:

  • Separate cytoskeletal and soluble fractions

  • Analyze FMN1 distribution between fractions using the antibody in Western blot

Background information indicates FMN1 co-localizes with the actin cytoskeleton and influences actin filament assembly, making this a physiologically relevant interaction to study .

How can I optimize FMN1-FITC antibody dilutions for different experimental applications?

Optimal antibody dilution varies by application and specific antibody product. Use this methodological approach to determine optimal dilutions:

• Initial dilution range testing:

  • For Western blotting: Test 1:300, 1:1000, 1:3000, and 1:5000 dilutions

  • For immunofluorescence: Test 1:50, 1:100, 1:200 dilutions

  • Include positive and negative controls for each dilution

• Signal-to-noise optimization:

  • Quantify specific signal intensity vs. background for each dilution

  • Calculate signal-to-noise ratio

  • Select dilution with highest ratio, not necessarily strongest absolute signal

• Application-specific considerations:

  • Western blotting typically requires less antibody (higher dilutions)

  • Immunofluorescence generally requires more concentrated antibody

  • Flow cytometry may require intermediate concentrations

These ranges are based on manufacturer recommendations for specific FMN1 antibody products .

How can surface plasmon resonance (SPR) be used with FMN1 antibodies to study protein-protein interactions?

Surface plasmon resonance provides real-time, label-free analysis of molecular interactions. When working with FMN1 antibodies:

• SPR experimental design for FMN1 studies:

  • Immobilize purified FNBP4 on sensor chip surface

  • Flow FMN1 protein domains (FH1-FH2 or FH2 alone) at various concentrations

  • Measure association and dissociation rates in real-time

  • Calculate binding affinity (KD)

• Sample preparation protocol:

  • Express and purify FMN1 domains (FH1-FH2, amino acids 870-1466; FH2, amino acids 983-1466)

  • Dialyze proteins against HBS-N buffer (0.01 M HEPES, 0.15 M NaCl, pH 7.4) for 4 hours at 4°C

  • Filter samples through 0.22 μm filters to remove aggregates

• Data analysis approach:

  • Fit sensorgrams to appropriate binding models (1:1 Langmuir, heterogeneous ligand, etc.)

  • Extract association (ka) and dissociation (kd) rate constants

  • Calculate equilibrium dissociation constant (KD = kd/ka)

Recent research has successfully applied SPR to characterize binding kinetics between FNBP4 and FMN1, demonstrating the utility of this approach for quantitative interaction studies .

What ELISA-based methods can be developed to quantify FMN1 using specific antibodies?

ELISA methods for FMN1 quantification:

• Sandwich ELISA protocol:

  • Coat 96-well ELISA plates (Maxisorp surface) with 10 μg purified capture antibody in PBS overnight at 4°C

  • Block with 5% BSA in PBS for 2 hours at room temperature

  • Incubate with sample containing FMN1 or purified FMN1 standards for 2 hours

  • Add detection antibody (e.g., FITC-conjugated anti-FMN1)

  • For FITC-conjugated antibodies, measure fluorescence directly; alternatively, use HRP-conjugated secondary antibody and colorimetric detection

  • Generate standard curve for quantification

• Competitive ELISA approach:

  • Pre-incubate samples with limiting amount of FITC-labeled anti-FMN1

  • Add to plates coated with recombinant FMN1

  • Measure inverse relationship between sample FMN1 concentration and FITC signal

• Detection of protein-protein interactions:

  • Coat ELISA plates with purified FNBP4

  • Add FH1-FH2 FMN1 or FH2 FMN1 protein at varying concentrations

  • Detect bound FMN1 using specific polyclonal antibodies

  • This approach has been validated for detecting FNBP4-FMN1 complexes

Research has demonstrated successful use of ELISA to study FMN1-FNBP4 interactions using mice anti-FMN1 antibodies at 1:1000 dilution .

How can FMN1 antibodies be incorporated into modern antibody screening technologies?

Incorporating FMN1 antibodies into advanced screening platforms:

• Genotype-phenotype linked screening:

  • Apply dual-expression vector systems based on Golden Gate cloning

  • Express membrane-bound FMN1 antibodies in vivo

  • Screen for high-affinity binders using flow cytometry

  • This approach can reduce screening time to approximately 7 days

• Next-generation sequencing integration:

  • Combine antibody repertoire analysis with functional screening

  • Identify FMN1-reactive antibody sequences from immunized animals

  • Express and validate candidates using recombinant approaches

• Single B-cell isolation technologies:

  • Isolate FMN1-reactive B cells using fluorescently labeled antigens

  • Perform single-cell RT-PCR to amplify antibody genes

  • Express recombinant antibodies for validation

Recent advances in antibody technology, including genotype-phenotype linked screening systems, have accelerated the development of high-affinity antibodies and could be applied to generate improved FMN1-specific antibodies .

What is the potential for using FMN1 antibodies in super-resolution microscopy techniques?

Applying FMN1-FITC antibodies in super-resolution microscopy:

• STED (Stimulated Emission Depletion) microscopy:

  • FITC fluorophores can be used in STED with appropriate depletion lasers

  • Resolution improvement from ~250 nm to ~50 nm enables detailed visualization of FMN1 localization at the cytoskeleton

  • Protocol adjustments: Increase antibody concentration by ~25% compared to standard IF

  • Mount in specialized STED-compatible mounting media

• STORM/PALM approaches:

  • For single-molecule localization microscopy, consider photoconvertible fluorophore conjugates

  • Alternatively, use secondary probes with appropriate photoswitching properties

  • These techniques can resolve FMN1 distribution at 10-20 nm resolution

• Expansion microscopy compatibility:

  • FITC antibodies are generally compatible with protein retention expansion microscopy

  • Physical expansion of specimens can provide 4-10× improvement in effective resolution

  • Protocol modification: Use lower concentration of antibody (1:300-1:500) to prevent background amplification

• Multicolor super-resolution:

  • Combine FMN1-FITC with spectrally distinct probes for cytoskeletal components

  • Enables nanoscale co-localization analysis of FMN1 with binding partners

These advanced imaging approaches could reveal previously unresolved details of FMN1 distribution and interactions with cytoskeletal components and binding partners .

What are emerging trends in FMN1 antibody applications for developmental biology research?

FMN1 antibodies are increasingly being applied in developmental biology research contexts:

• Lineage-specific expression analysis:

  • Tracking FMN1 expression during embryonic development using immunohistochemistry

  • Correlating expression patterns with developmental abnormalities

  • Investigating tissue-specific splicing variants using domain-specific antibodies

• Organoid applications:

  • Studying FMN1 distribution in 3D organoid cultures

  • Investigating cytoskeletal organization during organoid formation

  • Protocol considerations include extended antibody incubation times (24-48 hours) and specialized clearing techniques

• Mechanistic studies of limb development:

  • Investigating FMN1's role in Sonic hedgehog/FGF-4 feedback loops

  • Co-localization with signaling pathway components

  • Correlation of FMN1 distribution with cell polarity in developing limb buds

Background information indicates FMN1 plays crucial roles in limb development and kidney formation, with deficiency mutations leading to developmental defects. Future research may further elucidate the molecular mechanisms underlying these phenotypes .

How can researchers validate the specificity of new FMN1 antibody preparations?

Comprehensive validation strategy for FMN1 antibodies:

• Molecular validation:

  • Western blot analysis showing appropriate molecular weight (full-length human FMN1: ~1419 amino acids)

  • Immunoprecipitation followed by mass spectrometry identification

  • Peptide competition assays using the immunizing peptide

• Cellular validation:

  • Immunostaining patterns consistent with known subcellular locations (cytoplasm, nucleus, cell membrane)

  • siRNA knockdown or CRISPR knockout showing reduced or absent signal

  • Correlation with mRNA expression patterns across tissues

• Cross-reactivity assessment:

  • Testing against recombinant FMN1 from multiple species

  • Evaluation of potential cross-reactivity with related formins (FMN2, mDia1-3)

  • Epitope alignment analysis across species to predict cross-reactivity

• Application-specific validation:

  • For each intended application (WB, IF, IHC, etc.), include positive and negative controls

  • For FITC-conjugated antibodies, confirm fluorophore:protein ratio (typically 3-6 FITC molecules per antibody)

  • Assess lot-to-lot consistency when reordering