FAM53A Antibody, HRP conjugated

What is FAM53A and why is it significant 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 that plays an important role in neural development by specifying dorsal cell fates within the neural tube. It is highly expressed in the dorsal regions of the midbrain, hindbrain, diencephalon, and spinal neural tube, with lower expression levels in the branchial arches, telencephalon, heart, and somites of developing embryos . Recent studies have demonstrated that FAM53A affects breast cancer cell proliferation, migration, and invasion through the MEK-ERK pathway, with effects dependent on the p53 status of the cells .

Methodology for studying FAM53A expression includes:

• Immunohistochemical staining of tissue specimens

• Immunofluorescence microscopy for subcellular localization

• Western blotting for protein expression analysis

• siRNA knockdown for functional studies

What is the principle behind HRP conjugation to antibodies?

Horseradish peroxidase (HRP) conjugation to antibodies involves the chemical linking of the enzyme to antibody molecules through covalent bonds. The most common approach utilizes sodium meta periodate to generate aldehyde groups by oxidation of carbohydrate moieties on HRP. These activated aldehydes then combine with amino groups on antibodies to form Schiff's bases, which are subsequently stabilized through reduction with sodium cyanoborohydride .

The conjugation process typically follows these steps:

• Oxidation of HRP with sodium meta periodate

• Purification of activated HRP

• Mixing of activated HRP with antibodies (typically at 1 mg/ml concentration)

• Reduction of formed Schiff's bases

• Purification of the conjugate

An enhanced method involving lyophilization of activated HRP before antibody conjugation has demonstrated significantly improved sensitivity in immunoassay applications .

How should I store and handle HRP-conjugated FAM53A antibodies?

For optimal performance and longevity of HRP-conjugated antibodies:

• Store in light-protected vials or covered with a light-protecting material (e.g., aluminum foil)

• Conjugated antibodies are stable for at least 12 months at 4°C

• For longer storage (up to 24 months), dilute conjugates with up to 50% glycerol and store at -20°C to -80°C

• Avoid repeated freezing and thawing as this will compromise enzyme activity and antibody binding

• Most commercial HRP-conjugated antibodies are supplied in a buffer containing stabilizers (typically 0.01M TBS, pH 7.4, with 1% BSA, 0.02% Proclin300, and 50% Glycerol)

What are the recommended dilutions and applications for FAM53A antibody with HRP conjugation?

Based on manufacturer specifications and research protocols, the following applications and dilutions are recommended:

Note: Optimal dilutions should be determined by each laboratory for each specific application .

How can I validate the specificity of FAM53A antibody in my experiments?

To ensure the specificity and reliability of FAM53A antibody detection:

• Positive and negative controls:

  • Use cell lines with known FAM53A expression (e.g., MDA-MB-231 shows high expression, while MCF-7 shows lower expression)

  • Include non-malignant human mammary epithelial cells (MCF-10A) as reference

• Knockdown validation:

  • Perform siRNA knockdown of FAM53A (e.g., using sc-88998 siRNA)

  • Compare antibody staining in knockdown vs. control samples

• Immunofluorescence localization:

  • Verify nuclear and cytoplasmic localization pattern consistent with known distribution

  • Counterstain nuclei with DAPI to confirm subcellular localization

• Multiple detection methods:

  • Compare results from different techniques (Western blot, immunohistochemistry, immunofluorescence)

  • Verify band size (~44-53 kDa) in Western blot applications

How does the relationship between FAM53A and p53 status affect experimental design in cancer research?

The relationship between FAM53A and p53 is a critical consideration when designing experiments with FAM53A antibodies in cancer research. Studies have shown that FAM53A levels are negatively correlated with wild-type p53 status in breast cancer tissues . This relationship has significant implications:

Experimental considerations:

• Cell line selection: Choose cell lines with known p53 status:

  • p53 wild-type lines (e.g., MCF-7)

  • p53-null or mutant lines (e.g., MDA-MB-231, T47D, BT-549)

• Tissue sample stratification: When analyzing clinical samples, stratify by p53 status:

  p53 Status	FAM53A Expression	Observed Frequency	p-value
  Negative	Positive	53/115 (46.1%)	<0.001
  Negative	Negative	62/115 (53.9%)	<0.001
  Positive	Positive	17/84 (20.2%)	<0.001
  Positive	Negative	67/84 (79.8%)	<0.001

• Functional analysis: When studying FAM53A functions, consider that:

  • In p53 wild-type cells (MCF-7), FAM53A overexpression inhibits cell migration, invasion, and proliferation

  • In p53-null cells (MDA-MB-231), opposite effects are observed

  • These differences appear to be mediated through the MEK-ERK pathway

• Validation experiments: Include p53 manipulation experiments:

  • Silence TP53 in p53-positive cells

  • Express wild-type p53 in p53-negative cells

  • Use MEK inhibitors (e.g., PD98059) to verify pathway involvement

What are the methodological improvements in HRP-antibody conjugation that enhance immunoassay sensitivity?

Recent advances in HRP conjugation methodology have significantly improved the sensitivity of immunoassays using HRP-conjugated antibodies:

Enhanced conjugation protocol with lyophilization step:

• Oxidize HRP using sodium meta periodate to generate aldehyde groups

• Lyophilize the activated HRP (key modification)

• Mix the lyophilized activated HRP with antibodies (1 mg/ml)

• Complete conjugation with sodium cyanoborohydride reduction

Advantages of the modified protocol:

• Increased sensitivity: Conjugates prepared with the lyophilization step can detect antigens at dilutions of 1:5000, compared to 1:25 for classical methods (p<0.001)

• Lower detection limit: Conjugates can detect antigens as low as 1.5 ng

• Stability: Lyophilized activated HRP can be maintained at 4°C for longer durations

• Higher HRP incorporation: The lyophilization step reduces reaction volume without changing reactant amounts, resulting in more efficient conjugation based on collision theory

Comparative ELISA data:
The statistical analysis showed highly significant differences (p<0.001) between classical and modified conjugation methods across all dilutions tested in direct ELISA applications .

How can I optimize FAM53A detection in complex tissue samples like breast cancer?

Optimizing FAM53A detection in complex tissue samples requires careful consideration of sample preparation, antigen retrieval, and detection methodology:

• Sample preparation protocol:

  • Fix tissue in 10% neutral formalin

  • Embed in paraffin and cut into 4-μm thick sections

  • Bake sections at 70°C for 2 hours

  • Dewax in xylene, followed by rehydration through gradient alcohols to distilled water

• Antigen retrieval optimization:

  • Use 0.01M citrate buffer (pH 6.0) with high temperature and high pressure for 2 minutes

  • Block endogenous peroxidase activity with 0.3% hydrogen peroxide

  • Reduce non-specific binding with 5% normal goat serum for 30 minutes at 20°C

• FAM53A staining evaluation system:
  Use the Immune Response Score (IRS) calculation:

  • IRS = PP × SI

  • Where PP (percentage of positive cells): 0 (no dye), 1 (1-25%), 2 (26-50%), 3 (51-75%), 4 (76-100%)

  • And SI (staining intensity): 0 (no staining), 1 (weak), 2 (medium), 3 (strong)

  • Final scores range from 0-9; scores >3 classified as high FAM53A expression

• Controls and counterstaining:

  • Use nuclei counterstaining with hematoxylin

  • Include both positive controls (MDA-MB-231 cells) and negative controls (omitting primary antibody)

What are common issues with HRP-conjugated antibodies and how can they be addressed?

Common challenges with HRP-conjugated antibodies and their solutions include:

How can I determine whether my experimental results with FAM53A antibody are reliable?

To ensure the reliability of your experimental results with FAM53A antibody:

• Multiple detection methods validation:

  • Compare results from different techniques (Western blot, IHC, IF)

  • If possible, use antibodies targeting different epitopes of FAM53A

  • The most commonly used immunogen sequence for FAM53A antibodies is within amino acids 255-340/398 or the sequence "LDDLTCKAEAGPLQYSAETLNKSGRLFPLELNDQSPWKVFSGGPPVRSQAATGPDFSFLPGLSAAAHTMGLQWQPQSPRPGAGLGAASTVDPSEST"

• Functional validation:

  • Perform siRNA knockdown of FAM53A and confirm reduced antibody staining

  • Overexpress FAM53A and verify increased antibody signal

  • Correlate results with known biological functions (e.g., effects on cell migration, proliferation)

• p53 context consideration:

  • Interpret results in the context of p53 status

  • In p53-wild-type cells, FAM53A overexpression should inhibit cell migration, invasion, and proliferation

  • In p53-null cells, opposite effects should be observed

• Technical controls:

  • Include isotype control antibodies

  • Test for interference from endogenous biotin or peroxidase

  • Perform absorption controls when possible

How does FAM53A interact with the MEK-ERK pathway in different cellular contexts?

FAM53A has been shown to significantly affect the MEK-ERK pathway, but in a manner that depends on p53 status:

In p53 wild-type cells (MCF-7):

• FAM53A overexpression inhibits MEK and ERK phosphorylation

• This leads to downregulation of Snail, cyclin D1, RhoA, RhoC, and MMP9

• Concurrently, it upregulates E-cadherin and p21 expression levels

• The net effect is inhibition of cell migration, invasion, and proliferation

In p53-null cells (MDA-MB-231):

• FAM53A overexpression has opposite effects, promoting MEK and ERK phosphorylation

• This may enhance metastatic potential and cell proliferation

• These effects are abrogated by MEK inhibitor PD98059

Experimental evidence:

• The MEK inhibitor PD98059 (10 μM for 1 h in MCF-7 cells; 25 μM for 2 h in MDA-MB-231 cells) reduces the biological effects of FAM53A knockdown in MCF-7 cells and FAM53A overexpression in MDA-MB-231 cells

• Silencing TP53 in MCF-7 cells and stably expressing wild-type p53 in MDA-MB-231 cells confirmed that these effects depend on p53 status

This dual role suggests that FAM53A may be:

• A tumor suppressor in p53-positive breast cancer

• A potential oncogene in p53-negative breast cancer

• A candidate for targeted anticancer therapies in p53-negative breast cancer

What are the latest methodological approaches for studying FAM53A's roles in cancer progression?

Advanced methodological approaches for investigating FAM53A's roles in cancer include:

• Gene expression modulation techniques:

  • siRNA knockdown (e.g., sc-88998 from Santa Cruz Biotechnology)

  • Expression vectors for overexpression studies

  • CRISPR-Cas9 gene editing for knockout studies

  • Inducible expression systems to control timing of expression changes

• Cell-based functional assays:

  • Migration assays (wound healing/transwell)

  • Invasion assays using Matrigel

  • Proliferation assays (MTT, BrdU incorporation)

  • Colony formation assays

  • These assays should be performed in both p53-positive and p53-negative contexts

• Signaling pathway analysis:

  • Western blotting for phosphorylated MEK and ERK

  • Use of pathway inhibitors (e.g., PD98059 for MEK inhibition)

  • Analysis of downstream targets (Snail, cyclin D1, RhoA, RhoC, MMP9, E-cadherin, p21)

  • Co-immunoprecipitation to identify protein interaction partners

• Clinical correlation studies:

  • Immunohistochemical analysis of tissue microarrays

  • Correlation with clinicopathological parameters

  • Scoring systems like the Immune Response Score (IRS = PP × SI)

  • Analysis stratified by p53 status

What are emerging areas of research involving FAM53A antibodies in cancer and neurodevelopment?

Several promising research directions are emerging for FAM53A investigations:

• Therapeutic targeting in p53-negative cancers:

  • Development of inhibitors targeting FAM53A in p53-negative tumors

  • Combination therapies with MEK/ERK inhibitors

  • Personalized treatment approaches based on p53 and FAM53A status

• Biomarker development:

  • Validation of FAM53A as a prognostic or predictive biomarker in breast cancer

  • Development of liquid biopsy assays for circulating FAM53A

  • Multiplex immunoassays combining FAM53A with other markers

• Neurodevelopmental studies:

  • Investigation of FAM53A's role in neural tube development

  • Studies in neurodevelopmental disorders, particularly those affecting dorsal neural tube regions

  • Potential connections to Huntington's disease (the FAM53A gene is located on chromosome 4 near the Huntingtin gene)

• Molecular interaction networks:

  • Identification of FAM53A-interacting proteins beyond the MEK-ERK pathway

  • Exploration of potential post-translational modifications regulating FAM53A

  • Integration of FAM53A into broader signaling networks

How might innovations in antibody conjugation technology impact FAM53A research?

Emerging antibody conjugation technologies have the potential to significantly advance FAM53A research:

• Site-specific conjugation methods:

  • Engineered antibodies with incorporated unnatural amino acids for site-specific conjugation

  • Use of sortase-mediated conjugation for controlled attachment of HRP

  • These approaches may improve homogeneity and reduce impact on antigen binding

• Multiplexed detection systems:

  • Dual labeling with HRP and fluorophores for combined detection methods

  • Multiplexed immunoassays to simultaneously detect FAM53A and interacting proteins

  • Sequential imaging protocols to evaluate co-localization with multiple markers

• Enhanced stabilization techniques:

  • Development of conjugates stable at working concentrations as low as 0.5 μg/mL

  • Storage stability for extended periods (60+ days at 37°C)

  • Formulations allowing stable liquid storage without lyophilization

• Scalable conjugation approaches:

  • Fully scalable methods from small-scale research (0.01 mg) to large-scale production (gram scale)

  • Standardized protocols to ensure batch-to-batch consistency

  • These advances would facilitate more reproducible research and potential clinical applications