FAM53A Antibody, FITC conjugated

What is FAM53A and why is it a target of interest in research?

FAM53A (Family with sequence similarity 53-member A), also known as Dorsal neural-tube nuclear protein (DNTNP), is a 398 amino acid nuclear protein with significant roles in neural development. The protein is thought to play an important role in specifying dorsal cell fates within the neural tube. FAM53A is widely expressed in the dorsal neural tube, with highest expression in the dorsal regions of the midbrain, hindbrain, diencephalon, and spinal neural tube. Lower expression levels are found in the branchial arches, telencephalon, heart, and somites during embryonic development. The gene is located on chromosome 4, which also contains the Huntingtin gene associated with Huntington's disease. Recent research has demonstrated that FAM53A expression negatively correlates with p53 status in breast cancer and affects cell migration, invasion, and proliferation through the MEK-ERK pathway, highlighting its potential significance in cancer research .

What distinguishes a FITC-conjugated FAM53A antibody from unconjugated versions?

A FITC-conjugated FAM53A antibody has fluorescein isothiocyanate molecules chemically attached to the antibody structure, unlike unconjugated versions. This direct conjugation enables visualization of antibody-antigen binding without requiring secondary detection reagents. FITC conjugation allows for direct detection using fluorescence microscopy, flow cytometry, or other fluorescence-based techniques with appropriate excitation (approximately 495nm) and emission (approximately 525nm) filters. Unconjugated FAM53A antibodies, such as those available from commercial suppliers, require a secondary antibody conjugated to a detection molecule for visualization. The selection between conjugated and unconjugated antibodies depends on experimental design, desired sensitivity, and compatibility with other markers in multi-labeling experiments .

What are the typical specifications of commercially available FAM53A antibodies?

Commercial FAM53A antibodies typically share these specifications:

• Antibody Type: Primary

• Clonality: Polyclonal (most commonly from rabbit hosts)

• Isotype: IgG

• Reactivity: Human (some also react with dog and predicted to react with chicken)

• Applications: Western Blot, ELISA, immunohistochemistry (IHC-P, IHC-F), immunofluorescence (IF/IHC-P, IF/IHC-F, IF/ICC), and immunocytochemistry

• Target subcellular location: Nucleus

• Storage requirements: -20°C (avoid freeze-thaw cycles)

• Immunogen: Typically synthetic peptides from the C-terminal region or within the range of amino acids 255-340/398 of human FAM53A

• Storage buffer: Varies by manufacturer (examples include PBS with 2% sucrose or TBS with BSA and glycerol)

What are the spectral properties of FITC and how do they influence experimental design?

FITC (fluorescein isothiocyanate) exhibits specific spectral characteristics that researchers must consider when designing experiments:

• Excitation maximum: Approximately 495nm (blue light region)

• Emission maximum: Approximately 525nm (yellow-green light region)

• Quantum yield: High fluorescence efficiency when properly conjugated

• pH sensitivity: Optimal fluorescence at pH 8.0; significantly reduced at lower pH

• Photobleaching susceptibility: Relatively high compared to newer fluorophores

These properties influence experimental design in several ways:

• Microscopy: Requires appropriate filter sets (blue excitation, green emission)

• Flow cytometry: Typically detected in the FL1 channel

• Multiplexing considerations: Potential spectral overlap with PE (phycoerythrin) or other green-emitting fluorophores

• Buffer selection: Maintaining pH 7.2-8.0 for optimal signal

• Imaging parameters: Minimizing exposure time to reduce photobleaching

Understanding these properties allows researchers to optimize detection sensitivity and specificity when using FITC-conjugated FAM53A antibodies .

What is the optimal methodology for conjugating FITC to FAM53A antibodies?

The optimal methodology for conjugating FITC to FAM53A antibodies involves several critical parameters:

• Starting material quality:

  • Use highly purified FAM53A antibody (preferably via DEAE Sephadex chromatography)

  • Select high-quality FITC with verified reactivity

• Reaction conditions:

  • pH: 9.5 (optimal for the reaction of FITC with amino groups)

  • Temperature: Room temperature is typically sufficient

  • Protein concentration: 25 mg/ml (initial concentration)

  • Reaction time: 30-60 minutes (for maximal labeling)

  • Buffer: Carbonate or borate buffer (pH 9.5)

• Purification methods:

  • Gradient DEAE Sephadex chromatography separates optimally labeled antibodies from under- and over-labeled proteins

  • Gel filtration removes free FITC molecules

• Quality control:

  • Determine fluorescein/protein (F/P) ratio spectrophotometrically

  • Optimal F/P ratio ranges from 3-8 FITC molecules per antibody

  • Verify functionality through binding assays

This methodology ensures efficient conjugation while maintaining antibody binding activity and fluorescence properties .

How should researchers evaluate the quality of FITC-conjugated FAM53A antibodies?

Researchers should implement a comprehensive quality control process for FITC-conjugated FAM53A antibodies:

• Spectrophotometric analysis:

  • Measure absorbance at 280nm (protein) and 495nm (FITC)

  • Calculate F/P ratio using the formula:
    F/P = [A495 × molecular weight of IgG] / [195 × antibody concentration (mg/ml)]

  • Optimal F/P ratio: 3-8 FITC molecules per antibody

• Functionality testing:

  • Flow cytometry with positive control cells (known to express FAM53A)

  • Signal-to-noise ratio assessment using positive and negative cell populations

  • Competitive binding with unconjugated antibody

• Specificity validation:

  • Western blot correlation with FAM53A protein size (approximately 45 kDa)

  • Signal reduction in FAM53A knockdown samples

  • Peptide competition analysis

• Stability assessment:

  • Retain aliquots for testing fluorescence intensity over time

  • Monitor for precipitation or aggregation

  • Test after varying storage conditions

These evaluations ensure that FITC-conjugated FAM53A antibodies will perform reliably in research applications, providing specific detection with minimal background interference .

How can FITC-conjugated FAM53A antibodies be optimally used in flow cytometry?

Optimizing flow cytometry with FITC-conjugated FAM53A antibodies requires careful consideration of several methodological aspects:

• Sample preparation protocol:

  • Complete cell fixation (2-4% paraformaldehyde) to preserve structure

  • Permeabilization with 0.1-0.3% Triton X-100 or saponin for nuclear antigen access

  • Blocking with 2-5% serum or BSA to reduce non-specific binding

  • Incubation with optimized antibody concentration (determined by titration)

• Essential controls:

  • Unstained cells (for autofluorescence assessment)

  • Isotype-FITC control (matching concentration)

  • FMO (Fluorescence Minus One) for multi-color panels

  • Positive control (cell line with confirmed FAM53A expression)

  • Negative control (FAM53A knockdown cells or pre-blocking with immunizing peptide)

• Instrument settings:

  • Appropriate voltage settings for FITC detection channel

  • Compensation setup if using multiple fluorophores

  • Consistent PMT settings between experiments

• Analysis considerations:

  • Gating strategy that accounts for cell size/viability

  • Comparative analysis using median fluorescence intensity (MFI)

  • Quantification of percent positive cells using proper threshold setting

This methodological approach enables reliable detection and quantification of FAM53A expression across different cell populations or treatment conditions .

What experimental design is recommended for studying FAM53A in relation to the MEK-ERK pathway?

To effectively investigate FAM53A's relationship with the MEK-ERK pathway using FITC-conjugated antibodies, researchers should consider this experimental design:

• Cell model selection:

  • Include cell lines with varying baseline FAM53A expression

  • Create stable FAM53A overexpression and knockdown models

  • Consider p53 status (wild-type vs. mutant) based on known correlations

• MEK-ERK pathway modulation:

  • Pharmacological intervention using:

    • MEK inhibitors (PD98059, U0126)

    • ERK inhibitors (SCH772984)

    • Pathway activators (growth factors, phorbol esters)

  • Genetic manipulation of pathway components

• Readout methodologies:

  • Flow cytometry to quantify FAM53A expression changes

  • Multi-color immunofluorescence to co-localize FAM53A with:

    • Phospho-MEK

    • Phospho-ERK

    • Downstream transcription factors

  • Western blot for validation and quantification

• Time-course analysis:

  • Short-term responses (minutes to hours)

  • Long-term adaptations (days)

  • Recovery after inhibitor withdrawal

Research by Li et al. demonstrated that MEK inhibitor PD98059 reduced the biological effects of FAM53A in breast cancer cells, suggesting that comprehensive pathway analysis will yield valuable insights into FAM53A function .

What approaches can be used to investigate FAM53A localization and dynamics using FITC-conjugated antibodies?

Investigating FAM53A localization and dynamics with FITC-conjugated antibodies can be accomplished through several sophisticated approaches:

• Live-cell imaging techniques:

  • Microinjection of FITC-conjugated FAM53A antibodies

  • Combine with organelle markers for co-localization studies

  • Time-lapse microscopy following stimulation

• Advanced microscopy methods:

  • Confocal microscopy for high-resolution subcellular localization

  • Super-resolution microscopy (STED, STORM, PALM) for nanoscale distribution

  • FRAP (Fluorescence Recovery After Photobleaching) to study protein mobility

  • FRET (with appropriate acceptor fluorophore) to detect protein interactions

• Quantitative image analysis:

  • Nuclear/cytoplasmic intensity ratio measurements

  • Co-localization coefficients with other proteins

  • Tracking of intensity changes in response to stimuli

• Experimental manipulations to track dynamics:

  • Cell cycle synchronization to monitor cell-cycle-dependent changes

  • Drug treatments that affect nuclear transport

  • Stress conditions (oxidative stress, hypoxia)

  • p53 activation/inhibition based on known correlations

• Validation approaches:

  • Comparison with GFP-tagged FAM53A in transfection studies

  • Correlation with biochemical fractionation results

These methodologies enable comprehensive analysis of both static localization and dynamic behavior of FAM53A under various physiological and experimental conditions .

What are common technical challenges when using FITC-conjugated antibodies and their solutions?

Researchers frequently encounter several technical challenges when working with FITC-conjugated antibodies, including those targeting FAM53A:

These solutions help ensure consistent, specific detection of FAM53A with minimal artifacts or false positives .

How can researchers validate the specificity of FITC-conjugated FAM53A antibody staining?

Validating the specificity of FITC-conjugated FAM53A antibody staining requires a multi-faceted approach:

• Genetic manipulation controls:

  • Compare staining intensity in:

    • FAM53A knockdown cells (siRNA, shRNA)

    • FAM53A knockout cells (CRISPR-Cas9)

    • FAM53A overexpression systems

  • Signal should correlate with expression level

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Should result in significant signal reduction

  • Include gradient of competing peptide concentrations

• Cross-validation with multiple detection methods:

  • Compare FITC-conjugated antibody results with:

    • Unconjugated primary + secondary detection

    • Alternative FAM53A antibody targeting different epitope

    • mRNA expression data (in situ hybridization or qPCR)

• Western blot correlation:

  • Perform western blot using the same antibody

  • Confirm detection of protein at expected molecular weight (~45 kDa)

  • Compare expression levels across samples with flow cytometry results

• Subcellular localization verification:

  • Confirm nuclear localization pattern consistent with known biology

  • Co-stain with nuclear markers

• Isotype and secondary-only controls:

  • Include matched isotype-FITC control

  • For indirect detection, include secondary-only control

This systematic validation ensures that experimental observations accurately reflect FAM53A biology rather than artifacts or non-specific binding .

What strategies can improve detection sensitivity when FAM53A expression is low?

When FAM53A expression is low, researchers can employ several strategies to enhance detection sensitivity with FITC-conjugated antibodies:

• Signal amplification methods:

  • Tyramide Signal Amplification (TSA): Can increase sensitivity 10-100 fold

  • Avidin-biotin amplification systems

  • Polymer-based detection systems with multiple fluorophores

• Optimized sample preparation:

  • Enhanced antigen retrieval for fixed tissues (citrate or EDTA-based)

  • Extended primary antibody incubation (overnight at 4°C)

  • Optimized permeabilization for nuclear antigen access

• Instrumentation adjustments:

  • Flow cytometry: Increased voltage on FITC detector

  • Microscopy: Extended exposure time, increased gain

  • Confocal settings: Increased laser power, expanded pinhole

• Imaging processing enhancements:

  • Deconvolution algorithms

  • Background subtraction techniques

  • Maximum intensity projections from z-stacks

• Alternative conjugation strategies:

  • Higher F/P ratio (while avoiding quenching)

  • Consider brighter fluorophores (Alexa 488)

  • Quantum dot conjugation for exceptional brightness

• Biological manipulation:

  • Stress conditions that may upregulate FAM53A

  • Cell synchronization if expression is cell-cycle dependent

  • MEK inhibition, which has been shown to affect FAM53A levels

These approaches, used individually or in combination, can significantly improve detection of low-abundance FAM53A, enabling more sensitive analyses of its expression and function .

How can FITC-conjugated FAM53A antibodies be used to investigate the relationship between FAM53A and p53 status?

The negative correlation between FAM53A levels and p53 status in breast cancer presents an important research direction. FITC-conjugated FAM53A antibodies can be utilized in sophisticated experimental designs to investigate this relationship:

• Dual-parameter analysis in patient samples:

  • Flow cytometry with FITC-FAM53A and compatible p53 antibodies

  • Multi-color immunohistochemistry on tissue microarrays

  • Quantitative analysis correlating expression patterns

• Mechanistic investigation in cell models:

  • Compare FAM53A levels in isogenic cell lines differing only in p53 status:

    • p53 wild-type (MCF-7) versus knockout

    • p53-null (MDA-MB-231) versus p53-restored

  • Time-course analysis following p53 activation:

    • DNA damage induction (radiation, doxorubicin)

    • Nutlin-3a treatment (MDM2 inhibitor)

    • Temperature shift with temperature-sensitive p53 mutants

• Transcriptional regulation studies:

  • Flow cytometric analysis of FAM53A expression after:

    • p53 silencing (siRNA)

    • p53 overexpression

    • Expression of different p53 mutants

• MEK-ERK pathway intersection:

  • Three-color flow cytometry for FAM53A, p53, and phospho-ERK

  • Combine MEK inhibitors with p53 modulation

  • Track changes in cell behavior (migration, invasion, proliferation)

This approach would generate quantitative data on how FAM53A expression correlates with p53 status under various conditions, potentially revealing regulatory mechanisms and functional significance in cancer progression .

What considerations are important for designing multiplexed experiments that include FITC-conjugated FAM53A antibodies?

Designing effective multiplexed experiments with FITC-conjugated FAM53A antibodies requires careful consideration of several technical factors:

• Fluorophore selection and spectral compatibility:

  • FITC emission (525nm) creates constraints for panel design

  • Compatible fluorophores for 4-color panels include:

    • DAPI (blue, nuclear counterstain)

    • TRITC/PE (orange/red)

    • APC/Cy5 (far-red)

  • Consider spectral viewers to assess overlap

• Antibody technical compatibility:

  • Host species conflicts: Avoid multiple primaries from same species

  • Optimize blocking to prevent cross-reactivity

  • Sequential staining may be required for certain combinations

  • Validate each antibody individually before multiplexing

• Fixation and permeabilization balance:

  • FAM53A requires nuclear access (adequate permeabilization)

  • Other targets may require different conditions

  • Test compatibility of fixation methods for all targets

• Compensation and controls:

  • Single-stained controls for each fluorophore

  • FMO (Fluorescence Minus One) controls

  • Biological negative and positive controls for each target

• Acquisition and analysis considerations:

  • Sequential scanning to minimize crosstalk

  • Consistent voltage/gain settings between samples

  • Specialized software for colocalization analysis

  • Standardized quantification metrics

• Experimental validation:

  • Compare multiplexed results with single-staining

  • Assess potential antibody interference

  • Evaluate reproducibility across multiple experiments

These considerations ensure reliable, interpretable results from multiplexed experiments investigating FAM53A in relation to other proteins of interest .

How can researchers integrate FITC-conjugated FAM53A antibody data with other molecular and cellular analyses?

Integrating FITC-conjugated FAM53A antibody data with other molecular and cellular analyses creates a comprehensive understanding of FAM53A biology:

• Multi-omics integration approaches:

  • Correlate flow cytometry FAM53A protein levels with:

    • Transcriptomics (RNA-seq, microarray)

    • Proteomics (mass spectrometry)

    • Phosphoproteomics (for signaling pathway status)

    • Epigenomics (ChIP-seq, ATAC-seq)

  • Use computational tools for pathway analysis and network reconstruction

• Single-cell multi-parameter analysis:

  • Index sorting: FITC-FAM53A flow cytometry followed by:

    • Single-cell RNA-seq

    • Single-cell ATAC-seq

    • Clonal outgrowth and functional testing

  • Mass cytometry (CyTOF) with metal-tagged antibodies for extended panels

• Functional correlation methods:

  • Sort cells based on FAM53A-FITC intensity for:

    • Migration/invasion assays

    • Proliferation assessment

    • Drug response profiling

    • In vivo tumor formation (xenografts)

• Spatial context integration:

  • Correlate flow cytometry data with:

    • Spatial transcriptomics

    • Multiplex immunohistochemistry

    • Digital spatial profiling

• Temporal dynamics analysis:

  • Time-series experiments tracking FAM53A changes during:

    • Cell cycle progression

    • Differentiation processes

    • Response to treatment

    • Disease progression

This multi-dimensional approach allows researchers to position FAM53A within broader cellular networks and understand its functional relationships with other molecules, particularly in the context of the MEK-ERK pathway and p53 status in cancer research .