ATF5 Antibody, FITC conjugated

What is ATF5 and what cellular functions does it regulate?

ATF5 (also known as ATFX) is a cyclic AMP-dependent transcription factor belonging to the ATF/CREB family of transcription factors. It plays essential roles in several critical cellular processes, particularly in hematopoietic cell survival and neuronal cell differentiation . ATF5 functions primarily as a transcriptional regulator that influences cell cycle progression and apoptosis pathways. Research has demonstrated that ATF5 can upregulate cyclin D3 expression and activate anti-apoptotic mechanisms through the regulation of Bcl-2 and NFkB . In neural development, ATF5 acts as a negative regulator of differentiation, effectively blocking the differentiation of neuroprogenitor cells into neurons . This inhibitory function must be downregulated to permit normal neuronal differentiation to proceed.

What applications is the ATF5 Antibody, FITC conjugated suitable for?

The ATF5 Antibody, FITC conjugated, has been validated for ELISA applications according to manufacturer specifications . While ELISA is the primary validated application, the FITC conjugation makes this antibody particularly suitable for fluorescence-based detection methods including flow cytometry, immunofluorescence microscopy, and potentially fluorescence-activated cell sorting (FACS). The antibody specifically recognizes human ATF5 protein (UniprotID: Q9Y2D1) and has been generated using recombinant human cyclic AMP-dependent transcription factor ATF-5 protein (amino acids 1-282) as the immunogen .

What are the optimal storage conditions for ATF5 Antibody, FITC conjugated?

To maintain antibody integrity and functionality, the ATF5 Antibody, FITC conjugated should be stored at -20°C or -80°C upon receipt . Manufacturers specifically caution against repeated freeze-thaw cycles, which can damage the antibody structure and compromise its performance . The antibody is supplied in a liquid form in a buffer containing 0.03% Proclin 300 as a preservative, along with 50% glycerol and 0.01M PBS at pH 7.4 . When working with the antibody, it is advisable to aliquot it into smaller volumes upon first thaw to minimize freeze-thaw cycles for the stock solution.

How do I confirm the specificity of the ATF5 antibody in my experimental system?

Confirming antibody specificity is crucial for reliable research outcomes. For ATF5 Antibody, FITC conjugated, researchers should implement multiple validation approaches:

• Positive and negative controls: Include cells or tissues known to express ATF5 (such as certain lymphoid cells or neuroprogenitors) as positive controls, and those that do not express ATF5 as negative controls .

• Peptide competition assay: Pre-incubate the antibody with excess recombinant ATF5 protein (preferably the same immunogen used to generate the antibody, amino acids 1-282 of human ATF5) before application to your samples . Specific binding should be blocked in this assay.

• Knockdown validation: Compare staining in wild-type cells versus cells where ATF5 has been knocked down using siRNA or CRISPR-Cas9 methods.

• Cross-validation: Compare results with another ATF5 antibody raised against a different epitope or using a different detection method (such as RT-PCR for mRNA expression).

How can I use ATF5 Antibody, FITC conjugated in studies investigating apoptotic pathways?

ATF5 has been identified as an anti-apoptotic factor, particularly in hematopoietic cells, making the FITC-conjugated antibody valuable for investigating survival pathways . When designing experiments focused on apoptosis:

• Flow cytometry co-staining protocol: Combine ATF5-FITC antibody with markers of apoptosis such as Annexin V (with a non-competing fluorophore) to correlate ATF5 expression levels with apoptotic status at the single-cell level. Research has shown that ATF5 expression correlates with decreased cell death in lymphoid cells, which can be monitored using Annexin V labeling .

• Time-course experiments: Monitor ATF5 expression during apoptosis induction with agents like IL-3 withdrawal (in FL5.12 cells) or doxorubicin treatment . The existing literature demonstrates that ATF5 levels typically decrease in cell lines undergoing apoptosis, including FL5.12, HT-2, HeLa, and HepG-2 cells .

• Downstream target analysis: Assess the relationship between ATF5 expression and anti-apoptotic factors like Bcl-2 and NFκB, which have been shown to be upregulated in cells with high ATF5 expression . This can be accomplished through immunofluorescence co-staining or by flow cytometry.

• Quantification approach: Use mean fluorescence intensity (MFI) measurements from flow cytometry to quantitatively assess the correlation between ATF5 expression levels and apoptotic resistance in your experimental system.

What considerations should be made when using ATF5 Antibody in neuronal differentiation studies?

ATF5 has been shown to block the differentiation of neuroprogenitor cells into neurons , making it an important marker in neurodevelopmental research. When studying neuronal differentiation:

• Temporal expression analysis: Track ATF5 expression throughout the differentiation timeline using the FITC-conjugated antibody. ATF5 should show downregulation as differentiation progresses .

• Co-staining protocol: Combine ATF5-FITC antibody with neuronal lineage markers (using compatible fluorophores) to establish the relationship between ATF5 downregulation and acquisition of neuronal phenotypes.

• In situ versus dissociated culture analysis: When examining brain tissue sections, compare results from in situ hybridization for ATF5 mRNA with immunofluorescence using the FITC-conjugated antibody to confirm expression patterns .

• Manipulation validation: If experimentally altering ATF5 expression (overexpression or knockdown), use the antibody to confirm the effectiveness of your manipulation before assessing phenotypic outcomes.

• Microscopy optimization: For optimal imaging of FITC signal in brain tissue or neuronal cultures, implement appropriate antifade mounting media and control for autofluorescence in fixed neural tissues.

How can I design experiments to investigate ATF5's interaction with cyclin D3 promoter?

Research has established that ATF5 can activate cyclin D3 transcription, making the investigation of this interaction important for understanding ATF5's role in cell cycle regulation . A comprehensive experimental approach should include:

• Chromatin Immunoprecipitation (ChIP) protocol: While the FITC-conjugated antibody is not specifically validated for ChIP, parallel experiments could be designed using ChIP-validated ATF5 antibodies. Research has successfully employed HA-tag antibodies with HA-tagged ATF5 for this purpose . The basic protocol involves:

  • Crosslinking protein-DNA complexes in your cells of interest

  • Sonicating to shear chromatin

  • Immunoprecipitating with an appropriate ATF5 antibody

  • PCR amplification of cyclin D3 promoter regions containing putative ATF binding sites (TGACGTCA consensus sequence)

• Luciferase reporter assays: To functionally validate promoter interaction, implement luciferase reporter assays with constructs containing the cyclin D3 promoter with intact or mutated ATF binding sites. Previous research has demonstrated that ATF5 robustly activates cyclin D3 promoter in such assays .

• Comparative analysis: Include parallel experiments with related transcription factors like ATF4, which has been shown to be an even more potent activator of cyclin D3 than ATF5 in some contexts .

• Dominant-negative approach: Consider using dominant-negative forms of ATF5 (such as ATF5-DBD, which lacks the transactivation domain) as a control to confirm specificity of the interaction .

What controls should be included when studying ATF5's transcriptional activities?

When investigating ATF5's function as a transcription factor, particularly using the FITC-conjugated antibody for expression analysis:

• Related transcription factor controls: Include analysis of other ATF family members, particularly ATF4, which shares structural relatedness with ATF5 and may have overlapping functions .

• Promoter specificity controls: When examining transcriptional activation, include multiple promoters with ATF binding sites. Research shows ATF5 strongly activates cyclin D1 and D3 promoters but not cyclin A or E2F1 promoters despite the presence of ATF binding sites .

• Dominant-negative controls: Use dominant-negative versions of ATF5 (ATF5-DBD) to confirm specificity of transcriptional effects .

• Cell type controls: Compare ATF5 activity across different cell types, as its function may vary. For instance, ATF5 has different effects in lymphoid cells versus neuroprogenitor cells .

• Inducible expression systems: Consider using inducible expression systems (like those used with FL5.12-ATF5 cells) to demonstrate direct effects of ATF5 induction on target gene expression .

What is the optimal protocol for using ATF5 Antibody, FITC conjugated in flow cytometry?

While the ATF5 Antibody, FITC conjugated is primarily validated for ELISA , researchers can adapt it for flow cytometry with the following protocol:

• Cell preparation:

  • Harvest cells (1-5 × 10^6 cells per sample)

  • Wash twice with cold PBS containing 0.1% BSA

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature if intracellular staining is required

  • Permeabilize with 0.1% Triton X-100 in PBS for 5 minutes (ATF5 is a transcription factor requiring nuclear permeabilization)

• Blocking and staining:

  • Block with 5% normal serum from the same species as the secondary antibody (if additional antibodies are used) for 30 minutes

  • Dilute ATF5-FITC antibody appropriately (starting with manufacturer's recommended concentration, typically 1:100-1:500)

  • Incubate cells with diluted antibody for 30-60 minutes at room temperature in the dark

  • Wash 3 times with PBS containing 0.1% BSA

• Flow cytometry acquisition:

  • Resuspend cells in appropriate buffer

  • Analyze using flow cytometer with 488 nm laser excitation and appropriate filter for FITC detection (typically 530/30 nm)

  • Include unstained and isotype controls (rabbit IgG-FITC) for gating strategy development

  • Consider including a viability dye to exclude dead cells from analysis

• Optimization considerations:

  • Titrate antibody to determine optimal concentration

  • Test different fixation and permeabilization protocols if signal is weak

  • Include samples with known high ATF5 expression (such as certain lymphoid cells or cell lines transfected with ATF5) as positive controls

How can I design experiments to investigate ATF5's role in hematopoietic cell survival?

Based on research showing ATF5's importance in hematopoietic cell survival , a comprehensive experimental design should include:

• Expression analysis protocol:

  • Isolate primary hematopoietic cells from different lineages and differentiation stages

  • Perform flow cytometry using ATF5-FITC antibody alongside lineage markers

  • Compare ATF5 expression levels across normal versus apoptosis-induced conditions, such as:

    • IL-3 withdrawal in relevant cell lines (e.g., FL5.12)

    • Serum deprivation

    • Doxorubicin treatment

  • Correlate ATF5 levels with apoptotic markers and survival rates

• Functional manipulation approaches:

  • Overexpression: Generate cell lines with inducible ATF5 expression (similar to the FL5.12-ATF5 system described in the literature)

  • Knockdown/knockout: Use siRNA or CRISPR-Cas9 to reduce ATF5 expression

  • Dominant-negative interference: Express ATF5-DBD to competitively inhibit endogenous ATF5 function

  • Monitor effects on:

    • Cell survival during stress conditions

    • Expression of downstream targets like cyclin D3, Bcl-2, and NFκB

    • Cell cycle progression

• In vivo validation:

  • Consider approaches similar to the transgenic mouse model with ATF5 expression under lymphocytic-specific promoter control

  • Analyze resulting phenotypes in hematopoietic compartments

  • Use the FITC-conjugated ATF5 antibody for flow cytometry analysis of isolated cells

What are the considerations for using ATF5 Antibody in multiplex immunofluorescence studies?

When incorporating ATF5 Antibody, FITC conjugated into multiplex immunofluorescence studies:

• Spectral compatibility planning:

  • FITC has excitation/emission peaks at approximately 495/519 nm

  • Select additional fluorophores with minimal spectral overlap, such as:

    • PE or Texas Red (for red channel)

    • APC (for far-red channel)

    • Pacific Blue (for blue channel)

  • If using automated systems, program appropriate compensation settings

• Sequential staining protocol:

  • For co-localization studies with other nuclear factors, consider sequential staining:

    • Complete ATF5-FITC staining first

    • Fix again lightly (0.5% paraformaldehyde for 5 minutes)

    • Proceed with subsequent antibody staining

• Signal amplification options:

  • If FITC signal is weak, consider:

    • Tyramide signal amplification (TSA) compatible systems

    • Anti-FITC secondary antibodies conjugated to brighter fluorophores

    • Longer incubation times with the primary antibody

• Image acquisition settings:

  • Minimize exposure time to prevent photobleaching of FITC

  • Consider spectral unmixing for cleaner separation of fluorophores

  • Use appropriate negative controls for autofluorescence subtraction

• Target combination suggestions:

  • ATF5 + cyclin D3 (demonstrated relationship in research)

  • ATF5 + Bcl-2 or NFκB (downstream targets in hematopoietic cells)

  • ATF5 + neuronal differentiation markers (for studies on neuroprogenitor differentiation)

How do I interpret ATF5 expression patterns in relation to cell cycle progression?

Research has established connections between ATF5 and cell cycle regulators, particularly cyclin D3 . When analyzing ATF5 expression patterns:

What are potential confounding factors when analyzing ATF5 expression data?

Several factors can complicate the interpretation of ATF5 expression analysis:

• Cell type variability considerations:

  • ATF5 function varies across cell types; for example, it has different roles in hematopoietic cells versus neuroprogenitors

  • Expression patterns may be cell type-specific and context-dependent

  • Always include appropriate positive control cells with known ATF5 expression patterns

• Stress response effects:

  • As a transcription factor involved in stress responses, ATF5 expression may be altered by experimental conditions

  • Control for variables such as:

    • Cell density and confluency

    • Serum levels and growth factor availability

    • Hypoxia or oxidative stress

    • Cell harvesting and processing methods

• Technical considerations:

  • FITC fluorescence can be affected by:

    • pH changes in fixation or buffer solutions

    • Photobleaching during extended imaging

    • Cell autofluorescence, particularly in cells with high metabolic activity

  • Include appropriate controls for each of these factors

• Antibody specificity issues:

  • Cross-reactivity with related ATF family members (especially ATF4, which shares structural similarity)

  • Potential recognition of both ATF5 isoforms if multiple start sites are utilized

  • Validate findings with independent methods such as RT-PCR for ATF5 mRNA

How can contradictory findings about ATF5's role in different cell types be reconciled?

Research demonstrates that ATF5 can have seemingly contradictory functions in different cellular contexts . To reconcile these differences:

• Systematic comparative analysis approach:

  • Design experiments that directly compare ATF5 function across multiple cell types under identical conditions

  • Include at minimum:

    • Hematopoietic cells (where ATF5 promotes survival)

    • Neuroprogenitor cells (where ATF5 blocks differentiation)

    • Other relevant cell types from your research

• Downstream pathway mapping:

  • Identify cell-type-specific downstream targets of ATF5

  • In hematopoietic cells, focus on:

    • Cyclin D3 activation

    • Bcl-2 and NFκB regulation

  • In neuroprogenitors, examine:

    • Differentiation inhibitory pathways

    • Maintenance of progenitor state

• Context-dependent factor identification:

  • Investigate co-factors that may interact with ATF5 in different cell types

  • Examine potential post-translational modifications that could alter ATF5 function

  • Consider the availability of dimerization partners (ATF5 can form heterodimers)

• Experimental validation strategies:

  • Perform "cell type swapping" experiments:

    • Express neuroprogenitor-derived ATF5 in hematopoietic cells and vice versa

    • Determine if cell-of-origin impacts ATF5 function

  • Create chimeric proteins combining domains from ATF5 with other factors to identify functional regions responsible for context-specific effects