FAM234A Antibody, FITC conjugated

What is FAM234A protein and what cellular functions does it serve?

FAM234A, also known as ITFG3 (Integrin Alpha FG-GAP Repeat Containing 3), is a human protein with UniProt accession Q9H0X4. The protein is encoded by the ITFG3 gene (KEGG: hsa:83986) and belongs to the FAM234 protein family . While its complete function remains under investigation, FAM234A is believed to play roles in cellular adhesion processes and potentially in signaling pathways due to its structural motifs. Recent research suggests potential involvement in mitochondrial function regulation, though more comprehensive studies are needed to fully elucidate its biological significance. The protein contains FG-GAP repeats which are often associated with protein-protein interactions in various cellular contexts.

What are the key specifications of the FAM234A Antibody, FITC conjugated reagent?

The FAM234A Antibody, FITC conjugated is a polyclonal antibody raised in rabbits against the recombinant Human Protein FAM234A protein (specifically amino acids 115-251). The antibody demonstrates reactivity with human FAM234A protein and has been purified using Protein G chromatography to achieve >95% purity . The antibody is of IgG isotype and is provided in liquid form in a buffer containing 0.01 M PBS (pH 7.4), 0.03% Proclin-300, and 50% glycerol . Being conjugated with FITC (Fluorescein isothiocyanate), this antibody has excitation/emission wavelengths of 499/515 nm and is compatible with the 488 nm laser line commonly found in flow cytometers and fluorescence microscopes .

What controls should be included when using FAM234A Antibody, FITC conjugated in flow cytometry or immunofluorescence studies?

A comprehensive experimental design using FAM234A Antibody, FITC conjugated should include multiple controls:

• Isotype Control: Include a FITC-conjugated rabbit IgG antibody at the same concentration to assess non-specific binding.

• Negative Cell Control: Use cell lines known not to express FAM234A protein.

• Blocking Control: Pre-incubate the antibody with recombinant FAM234A protein (ideally the immunogen fragment covering amino acids 115-251) to confirm specificity.

• Unstained Control: Include samples without any antibody to establish autofluorescence baseline.

• Single-Color Controls: When performing multi-parameter analysis, include single-stained samples for compensation.

These controls will collectively facilitate accurate interpretation of results, particularly in distinguishing specific staining from background or non-specific signals.

How should I determine the optimal working dilution for this antibody?

While the manufacturer indicates that optimal dilutions should be determined by the end user , a systematic titration approach is recommended:

• Prepare a series of antibody dilutions (typically 1:50, 1:100, 1:200, 1:500, 1:1000).

• Apply these dilutions to positive control samples (cells known to express FAM234A).

• Analyze signal-to-noise ratio for each dilution by comparing:

  • Mean fluorescence intensity (MFI) of positive cells

  • MFI of negative cells or isotype control

• Calculate the staining index: (MFI positive - MFI negative)/2 × standard deviation of MFI negative

• Select the dilution providing the highest staining index while minimizing background.

Initial tests should be performed with fresh aliquots, as antibody performance can be affected by storage conditions and freeze/thaw cycles.

What sample preparation protocols are compatible with FAM234A Antibody, FITC conjugated?

For optimal results with FAM234A Antibody, FITC conjugated, consider these preparation guidelines:

For Flow Cytometry:

• Harvest cells (typically 1×10^6 cells per sample)

• Wash twice with PBS containing 1% BSA

• For intracellular staining (likely needed for FAM234A):

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

  • Permeabilize with 0.1% Triton X-100 or 0.1% saponin in PBS for 15 minutes

• Block with 5% normal goat serum for 30 minutes

• Incubate with optimized dilution of FAM234A Antibody, FITC conjugated for 45-60 minutes at room temperature in the dark

• Wash three times with PBS/BSA

• Resuspend in appropriate buffer for analysis

For Immunofluorescence:

• Fix cells/tissue sections with 4% paraformaldehyde

• Permeabilize with 0.2% Triton X-100

• Block with 5-10% normal serum and 1% BSA

• Incubate with antibody dilution overnight at 4°C or 1-2 hours at room temperature

• Wash thoroughly with PBS

• Counterstain nucleus with DAPI if desired

• Mount using anti-fade mounting medium

Beyond standard applications, can FAM234A Antibody, FITC conjugated be used in multiplex immunofluorescence studies?

FAM234A Antibody, FITC conjugated can be effectively incorporated into multiplex immunofluorescence studies with careful panel design. The FITC fluorophore (excitation/emission: 499/515 nm) is compatible with standard filter sets and the 488 nm laser line . When designing multiplex panels:

• Select complementary fluorophores with minimal spectral overlap (e.g., PE, APC, Pacific Blue)

• Include appropriate compensation controls

• Consider the following compatibility table for common fluorophores:

• When examining subcellular co-localization, select markers with distinct cellular distributions

• Account for potential antibody cross-reactivity if using multiple rabbit-derived antibodies

This approach enables simultaneous assessment of FAM234A expression alongside other proteins of interest to investigate potential functional relationships.

How can I validate the specificity of FAM234A Antibody, FITC conjugated in my experimental system?

To rigorously validate the specificity of FAM234A Antibody, FITC conjugated:

• Genetic Validation:

  • Perform siRNA/shRNA knockdown of FAM234A in positive cell lines

  • Compare staining patterns between knockdown and control cells

  • Expect significant reduction in signal in knockdown samples

• Competitive Blocking:

  • Pre-incubate antibody with excess recombinant FAM234A protein (115-251 AA region)

  • Apply to samples in parallel with non-blocked antibody

  • Specific staining should be abolished or significantly reduced

• Orthogonal Detection Methods:

  • Confirm expression using alternative antibodies targeting different epitopes

  • Correlate protein detection with mRNA expression (RT-qPCR)

  • Western blot analysis (using non-conjugated version of the antibody)

• Multi-species Analysis:

  • The antibody is specified for human reactivity

  • Test on cells from different species to confirm specificity

  • Cross-reactivity information provides insights into epitope conservation

Thorough validation ensures confidence in subsequent experimental findings and facilitates accurate interpretation of results.

What approaches can resolve weak signal issues when working with FAM234A Antibody, FITC conjugated?

When encountering weak signals with FAM234A Antibody, FITC conjugated, consider these methodological adjustments:

• Antibody Concentration Optimization:

  • Increase antibody concentration (while monitoring background)

  • Extended incubation time (overnight at 4°C rather than 1 hour at room temperature)

• Sample Preparation Enhancement:

  • Optimize fixation protocol (test different fixatives and durations)

  • Explore alternative permeabilization reagents (Triton X-100, saponin, methanol)

  • Extend blocking time to reduce background and improve signal-to-noise ratio

• Signal Amplification Strategies:

  • Apply tyramide signal amplification (TSA) system

  • Use anti-FITC secondary antibody conjugated to a brighter fluorophore

  • Employ biotin-streptavidin amplification systems

• Instrumentation Adjustments:

  • Increase detector voltage/gain (flow cytometry) or exposure time (microscopy)

  • Verify filter sets match FITC excitation/emission characteristics (499/515 nm)

  • Ensure laser alignment and optimal instrument performance

• Biological Considerations:

  • Verify expression levels in your cell type (FAM234A may be expressed at low levels)

  • Consider stimulation conditions that might upregulate target expression

  • Check protein localization to ensure appropriate permeabilization

What quantitative approaches are recommended for analyzing FAM234A expression data?

Quantitative analysis of FAM234A expression using the FITC-conjugated antibody depends on the experimental platform:

For Flow Cytometry Data:

• Calculate percent positive cells using appropriate gating strategies:

  • Set gates based on isotype or FMO (Fluorescence Minus One) controls

  • Use histogram subtraction to isolate specific staining

• Quantify expression intensity:

  • Mean/Median Fluorescence Intensity (MFI)

  • Ratio of sample MFI to control MFI

  • Molecules of Equivalent Soluble Fluorochrome (MESF) calibration

• Statistical analysis:

  • For multiple samples, apply appropriate statistical tests (t-test, ANOVA)

  • Generate dose-response curves when examining treatments affecting FAM234A

For Immunofluorescence Microscopy:

• Measure signal intensity:

  • Integrated density measurements of defined cellular regions

  • Background subtraction using adjacent negative regions

  • Z-score normalization across multiple fields

• Analyze subcellular distribution:

  • Colocalization coefficients with organelle markers (Pearson's, Mander's)

  • Distance measurements from cellular landmarks

• Population analysis:

  • Single-cell intensity distribution to identify subpopulations

  • Correlation of expression with morphological features

These quantitative approaches enable robust comparison across experimental conditions and correlation with biological outcomes.

How should I interpret potential cross-reactivity in experiments using FAM234A Antibody, FITC conjugated?

When evaluating potential cross-reactivity with FAM234A Antibody, FITC conjugated:

Cross-reactivity assessment is particularly important when studying poorly characterized proteins like FAM234A, where knowledge of expression patterns and protein family similarities is still evolving.

How can FAM234A Antibody, FITC conjugated be utilized in live cell imaging studies?

While the antibody is primarily designed for fixed cell applications, live cell imaging with FAM234A Antibody, FITC conjugated requires specialized approaches:

• Membrane-associated FAM234A studies:

  • If FAM234A has extracellular domains, the antibody can potentially bind without permeabilization

  • Use physiological buffers (HBSS with Ca²⁺/Mg²⁺) supplemented with 2% FBS

  • Incubate cells with antibody (1:50-1:100 dilution) for 30-45 minutes at 4°C

  • Wash gently and image immediately to minimize internalization

• Intracellular delivery strategies:

  • Microinjection of antibody directly into cells

  • Protein transfection reagents (Chariot, ProJect)

  • Cell-penetrating peptide conjugation techniques

  • Electroporation with optimized parameters for cell viability

• Live imaging considerations:

  • Use temperature-controlled stage (37°C) with CO₂ regulation

  • Minimize phototoxicity with reduced laser power and interval imaging

  • Consider photobleaching characteristics of FITC when designing experiments

  • Use antifade reagents compatible with live cells (e.g., ProLong Live)

• Alternative approaches:

  • Generate FAM234A-fluorescent protein fusions (GFP, mCherry)

  • Use genetically encoded tags (SNAP, CLIP) with cell-permeable fluorescent ligands

  • Consider nanobody-based detection systems for improved intracellular delivery

These approaches enable dynamic studies of FAM234A trafficking, interactions, and responses to cellular stimuli that complement fixed-cell analyses.

What considerations are important when studying FAM234A in disease models or patient samples?

When investigating FAM234A in disease contexts using the FITC-conjugated antibody:

These methodological considerations enable robust investigation of FAM234A's potential roles in pathological processes and its utility as a biomarker.

What methodological approaches can elucidate FAM234A's potential role in cellular signaling pathways?

To investigate FAM234A's involvement in signaling networks using the FITC-conjugated antibody:

• Stimulation-response experiments:

  • Treat cells with pathway activators/inhibitors

  • Monitor FAM234A expression, localization, and post-translational modifications

  • Time-course experiments to capture dynamic changes

  • Recommended stimulants to test:

    • Growth factors (EGF, PDGF, IGF-1)

    • Cytokines (TNF-α, IL-6)

    • Stress inducers (oxidative stress, heat shock)

• Co-localization studies:

  • Perform dual labeling with markers for:

    • Signaling complexes (receptors, adaptor proteins)

    • Subcellular compartments (plasma membrane, endosomes, mitochondria)

    • Post-translational modification sites (phosphorylation, ubiquitination)

  • Use high-resolution techniques (confocal, STED, STORM) for precise localization

• Functional perturbation analysis:

  • Combine antibody detection with genetic manipulation:

    • FAM234A overexpression or knockdown

    • Mutation of key domains or residues

  • Assess impact on downstream signaling events

  • Correlate with cellular functions (proliferation, migration, differentiation)

• Interaction network mapping:

  • Proximity ligation assays to detect protein-protein interactions in situ

  • Immunoprecipitation followed by mass spectrometry

  • Computational prediction of interaction partners based on structural features

These approaches can systematically uncover FAM234A's functional roles in cellular signaling, contributing to a more comprehensive understanding of this protein's biological significance.