JUN (Ab-239) Antibody

What is c-Jun(Ab-239) Antibody and what epitope does it recognize?

c-Jun(Ab-239) Antibody is a rabbit polyclonal antibody that recognizes the peptide sequence around amino acids 237-241 (G-E-T-P-P) derived from Human c-Jun protein. This antibody is designed to detect endogenous levels of total c-Jun protein across multiple species including human, mouse, and rat . The target protein, c-Jun, functions as a transcription factor that recognizes and binds to the enhancer heptamer motif 5'-TGA[CG]TCA-3', playing critical roles in cellular signaling pathways . The antibody has been purified through affinity chromatography using epitope-specific peptide, ensuring high specificity for research applications .

What are the validated applications for c-Jun(Ab-239) Antibody?

The c-Jun(Ab-239) Antibody has been validated for Western Blotting (WB) and Immunohistochemistry (IHC) applications . For Western blotting applications, the recommended dilution range is 1:500 to 1:1000, while for immunohistochemistry applications, a dilution range of 1:50 to 1:100 is recommended . The predicted molecular weight of the c-Jun protein is approximately 43 kDa, which serves as a reference point when analyzing Western blotting results . These applications enable researchers to detect and quantify c-Jun expression in cell and tissue samples, facilitating studies on transcription factor dynamics and signaling pathways.

How should c-Jun(Ab-239) Antibody be stored and handled to maintain optimal activity?

For optimal preservation of antibody activity, c-Jun(Ab-239) Antibody should be stored at -20°C for long-term preservation, which is the manufacturer's recommended storage condition . For short-term use (within 6 months), the antibody can be stored at 4°C . The antibody is supplied at a concentration of 1.0mg/mL in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, containing 150mM NaCl, 0.02% sodium azide, and 50% glycerol . Avoiding repeated freeze-thaw cycles is critical for maintaining antibody integrity and performance. When working with the antibody, it should be kept on ice or at 4°C to minimize degradation during experimental procedures.

What are the recommended protocols for Western blotting using c-Jun(Ab-239) Antibody?

To achieve optimal results with c-Jun(Ab-239) Antibody in Western blotting, follow this methodological approach:

• Sample preparation: Extract total protein from cells or tissues using a suitable lysis buffer containing protease inhibitors.

• Protein separation: Load 20-50 μg of protein per lane on a 10-12% SDS-PAGE gel; c-Jun has a predicted molecular weight of 43 kDa.

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane using standard protocols.

• Blocking: Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute c-Jun(Ab-239) Antibody at 1:500 to 1:1000 in blocking buffer and incubate overnight at 4°C .

• Washing: Wash membrane 3-5 times with TBST.

• Secondary antibody: Incubate with HRP-conjugated anti-rabbit IgG secondary antibody.

• Detection: Develop using ECL substrate and capture images.

For phosphorylated c-Jun detection, consider phosphatase inhibitors in your lysis buffer and BSA rather than milk for blocking to avoid interference with phospho-epitopes.

What controls should be included when using c-Jun(Ab-239) Antibody in experimental workflows?

When designing experiments with c-Jun(Ab-239) Antibody, incorporate these essential controls:

• Positive control: Include cell lines with known c-Jun expression (e.g., HeLa cells stimulated with PMA or UV treatment to increase c-Jun levels).

• Negative control: Use cell lines with minimal c-Jun expression or c-Jun knockout cells.

• Technical controls:

  • Primary antibody omission control to assess non-specific binding of secondary antibody

  • Isotype control (rabbit IgG at the same concentration) to identify non-specific binding

  • Peptide competition assay using the immunizing peptide (G-E-T-P-P sequence) to confirm specificity

  • Loading control antibody (e.g., β-actin, GAPDH) to ensure equal protein loading

• Validation controls: For new experimental systems, confirm antibody specificity via siRNA/shRNA knockdown of c-Jun or CRISPR/Cas9 knockout samples.

These controls help distinguish true signals from artifacts and validate experimental findings.

How can c-Jun(Ab-239) Antibody be effectively used in immunohistochemistry applications?

For successful immunohistochemistry with c-Jun(Ab-239) Antibody, follow this methodological approach:

• Tissue preparation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues: Cut sections at 4-6 µm thickness

  • Frozen tissues: Cut sections at 6-8 µm thickness

• Antigen retrieval (for FFPE tissues):

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Microwave for 10-20 minutes or pressure cooker for 3-5 minutes

• Blocking:

  • Block endogenous peroxidase with 0.3% H₂O₂ in methanol for 10 minutes

  • Block non-specific binding with 5-10% normal serum from the same species as the secondary antibody

• Primary antibody incubation:

  • Dilute c-Jun(Ab-239) Antibody at 1:50-1:100 in antibody diluent

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

• Detection system:

  • Use a suitable detection system (e.g., ABC, polymer-based)

  • Develop with DAB or other appropriate chromogen

  • Counterstain with hematoxylin

• Controls:

  • Include positive control tissues known to express c-Jun

  • Include negative control sections with primary antibody omitted

Expected results: Nuclear staining pattern in positive cells, as c-Jun is a transcription factor primarily localized to the nucleus.

What are common issues encountered with c-Jun(Ab-239) Antibody in Western blotting and how can they be resolved?

For phosphorylated c-Jun detection, use phosphatase inhibitors in your lysis buffer and consider treating cells with stress inducers (UV, TNF-α, etc.) to increase phosphorylation states.

How can researchers distinguish between c-Jun and other related Jun family proteins when using c-Jun(Ab-239) Antibody?

The c-Jun(Ab-239) Antibody targets a specific peptide sequence around amino acids 237-241 (G-E-T-P-P) of c-Jun . To differentiate c-Jun from related Jun family proteins:

• Sequence alignment analysis:

  • Perform a sequence alignment of the epitope region (aa.237-241) across Jun family proteins (c-Jun, JunB, JunD)

  • The G-E-T-P-P sequence is relatively specific to c-Jun

• Experimental validation:

  • Run lysates from cells overexpressing individual Jun family members

  • Include recombinant Jun family proteins as reference standards

  • Perform siRNA knockdown of specific Jun family members to identify band specificity

• Molecular weight discrimination:

  • c-Jun: ~43 kDa

  • JunB: ~39 kDa

  • JunD: ~35 kDa (though multiple isoforms exist)

• Advanced validation:

  • Immunoprecipitation followed by mass spectrometry

  • Chromatin immunoprecipitation (ChIP) with promoter-specific primers

  • Co-immunoprecipitation with known specific interaction partners

If cross-reactivity remains a concern, consider using multiple antibodies targeting different epitopes and correlating the results.

How can c-Jun(Ab-239) Antibody be applied in protein-protein interaction studies involving AP-1 complexes?

c-Jun(Ab-239) Antibody can be valuable for investigating protein-protein interactions involving AP-1 complexes through these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use c-Jun(Ab-239) Antibody to precipitate c-Jun and its interacting partners

  • Protocol optimization: For nuclear transcription factors like c-Jun, use nuclear extraction buffers containing 400-450 mM NaCl for efficient extraction

  • Analyze co-precipitated proteins (Fos family members, ATF family proteins) by Western blotting

  • Recommended antibody amount: 2-5 μg per 500 μg of nuclear protein extract

• Chromatin Immunoprecipitation (ChIP):

  • Use c-Jun(Ab-239) Antibody to identify genomic binding sites of c-Jun

  • Optimize crosslinking conditions (1% formaldehyde for 10 minutes at room temperature)

  • Analyze c-Jun binding to known AP-1 target gene promoters

  • Combine with sequencing (ChIP-seq) for genome-wide binding profile analysis

• Proximity Ligation Assay (PLA):

  • Detect in situ protein-protein interactions between c-Jun and other transcription factors

  • Combine c-Jun(Ab-239) Antibody with antibodies against potential interaction partners

  • Visualize specific interactions as fluorescent spots under microscopy

• Bimolecular Fluorescence Complementation (BiFC):

  • Complement with plasmid-based expression systems to verify direct interactions

  • Can validate findings from antibody-based approaches

These approaches can reveal dynamic regulatory mechanisms of AP-1 complexes in various cellular contexts, particularly in stress response and oncogenic signaling pathways.

What are the considerations for using c-Jun(Ab-239) Antibody in phosphorylation-dependent studies of c-Jun activation?

c-Jun activity is heavily regulated by phosphorylation, particularly at serines 63/73 and threonines 91/93. When using c-Jun(Ab-239) Antibody in phosphorylation studies:

• Epitope accessibility considerations:

  • The epitope (aa.237-241) is distant from major phosphorylation sites, making this antibody suitable for detecting total c-Jun regardless of phosphorylation state

  • Use in parallel with phospho-specific c-Jun antibodies to calculate phosphorylation/total protein ratios

• Sample preparation for phosphorylation studies:

  • Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) in lysis buffers

  • Avoid phosphatase-rich tissues/cells without adequate inhibition

  • Process samples rapidly at 4°C to preserve phosphorylation states

• Stimulus-response experimental design:

  • Baseline: Serum-starve cells for 12-24 hours to reduce basal phosphorylation

  • Stimulation: Treat with UV (40 J/m²), TNF-α (10 ng/ml), EGF (100 ng/ml), or PMA (100 nM)

  • Time course: Collect samples at multiple timepoints (0, 15, 30, 60, 120 min)

• Data interpretation framework:

  • Compare phosphorylated/total c-Jun ratios rather than absolute phosphorylation levels

  • Consider JNK inhibitors (SP600125) as negative controls

  • Correlate phosphorylation with functional readouts (e.g., AP-1 reporter assays)

This approach enables mechanistic insights into c-Jun activation dynamics in diverse cellular contexts.

How can c-Jun(Ab-239) Antibody be adapted for use in advanced imaging techniques for studying transcription factor dynamics?

c-Jun(Ab-239) Antibody can be adapted for various advanced imaging techniques to study the spatial and temporal dynamics of c-Jun:

• Super-resolution microscopy (e.g., STED, STORM, PALM):

  • Secondary labeling: Use highly-specific fluorophore-conjugated secondary antibodies

  • Sample preparation: Optimize fixation with 4% PFA for 10 minutes at room temperature

  • Mounting: Use specialized anti-fade mounting media with refractive index matching

  • Expected resolution: 20-50 nm resolution of nuclear c-Jun distribution patterns

  • Controls: Include nuclear envelope markers for reference

• Live-cell imaging adaptations:

  • Primary approach: Cannot use antibody directly; complement with fluorescent protein-tagged c-Jun

  • Validation approach: Correlate live-cell dynamics with fixed cell antibody staining at multiple timepoints

  • Applications: Study c-Jun nuclear translocation in response to stimuli in real-time

• Fluorescence Recovery After Photobleaching (FRAP):

  • Application: Study c-Jun binding dynamics to chromatin

  • Method: Complement antibody validation with FRAP experiments using fluorescent protein-tagged c-Jun

  • Parameters: Measure t½ recovery to quantify c-Jun residence time on chromatin

• Immunoelectron microscopy:

  • Fixation: Glutaraldehyde (0.5-2%) with paraformaldehyde (2-4%)

  • Secondary labeling: Gold-conjugated secondary antibodies (different sizes for multi-protein localization)

  • Applications: Ultra-structural localization of c-Jun relative to nuclear substructures

  • Expected results: Visualization of c-Jun enrichment at euchromatin regions

These advanced imaging approaches can provide unprecedented insights into c-Jun's spatial organization and dynamic interactions within the nuclear microenvironment.

How can c-Jun(Ab-239) Antibody be incorporated into site-specific conjugation strategies for advanced research applications?

Leveraging modern antibody engineering approaches, c-Jun(Ab-239) Antibody can be modified through various site-specific conjugation strategies:

• Cysteine-based conjugation:

  • Approach: Reduction of interchain disulfide bonds followed by controlled conjugation with maleimide-functionalized molecules

  • Applications: Fluorophore conjugation for super-resolution microscopy

  • Advantages: Relatively straightforward chemistry without antibody engineering

  • Considerations: Maintain 1:1 stoichiometry to preserve antibody functionality

• Engineered tags for site-specific modifications:

  • Strategy: Express recombinant versions of the antibody with enzymatic tags (SNAP, CLIP, HaloTag)

  • Applications: Generate homogeneous antibody conjugates for quantitative imaging

  • Advantage: Precise control over the conjugation site

  • Limitation: Requires antibody sequence and recombinant production capabilities

• Unnatural amino acid incorporation:

  • Advanced approach: Insert azido-modified amino acids at specific positions

  • Chemistry: Click chemistry (copper-catalyzed or strain-promoted azide-alkyne cycloaddition)

  • Applications: Conjugation with bioorthogonal probes for live-cell imaging or pull-down experiments

  • Considerations: May induce immunogenicity or affect antibody folding

• Enzymatic approaches:

  • Method: Utilize enzyme-assisted ligation using formyl glycine-generating enzyme (FGE) or transglutaminase (TG)

  • Applications: Creation of homogeneous antibody-drug conjugates or imaging probes

  • Advantages: High specificity without disturbing antibody structure

  • Limitations: Potential immunogenicity from modified amino acid sequences

These site-specific conjugation strategies enable precise control over the antibody's functionality for specialized research applications beyond standard immunoassays.

What strategies can be employed to optimize c-Jun(Ab-239) Antibody for use in multi-parameter flow cytometry?

Optimizing c-Jun(Ab-239) Antibody for flow cytometry requires specific adaptations due to its nuclear target:

• Cell preparation protocol:

  • Fixation: 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilization: Use saponin (0.1%) for reversible permeabilization or methanol (-20°C) for robust nuclear antigen exposure

  • Nuclear permeabilization enhancers: Include 0.1% Triton X-100 to ensure nuclear membrane permeability

  • Buffer system: PBS with 1% BSA and 0.05% sodium azide

• Antibody titration strategy:

  • Start with manufacturer's recommended concentration (1:50-1:100)

  • Create a dilution series (1:25, 1:50, 1:100, 1:200, 1:400)

  • Evaluate signal-to-noise ratio for each concentration

  • Select optimal dilution based on separation index between positive and negative populations

• Multiparameter panel design:

  • Fluorophore selection: Choose bright fluorophores (PE, APC) for nuclear transcription factors

  • Compensation controls: Single-stained controls for each parameter

  • FMO (Fluorescence Minus One) controls: Especially important for nuclear antigens

  • Multiplexing approach: Combine with surface markers (CD markers) and other intracellular signaling markers

• Data analysis considerations:

  • Gating strategy: Exclude doublets and dead cells before analyzing c-Jun signal

  • Quantification method: Report median fluorescence intensity (MFI) rather than percent positive

  • Statistical approach: Use stimulation index (ratio of stimulated/unstimulated MFI)

This approach allows for simultaneous analysis of c-Jun expression with other cellular parameters in heterogeneous cell populations.

How can researchers design experiments using c-Jun(Ab-239) Antibody to study c-Jun's role in chromatin remodeling and epigenetic regulation?

To investigate c-Jun's involvement in chromatin remodeling and epigenetic regulation:

• ChIP-seq experimental design:

  • Cell preparation: Treat cells with relevant stimuli (e.g., TPA, UV, growth factors)

  • Crosslinking: 1% formaldehyde for 10 minutes at room temperature

  • Sonication: Optimize to achieve 200-500 bp DNA fragments

  • Immunoprecipitation: Use 5 μg c-Jun(Ab-239) Antibody per ChIP reaction

  • Controls: Input DNA and IgG immunoprecipitation

  • Analysis: Identify c-Jun binding sites and associated genes

• Sequential ChIP (ChIP-reChIP) protocol:

  • First IP: Use c-Jun(Ab-239) Antibody

  • Second IP: Use antibodies against histone modifications (H3K27ac, H3K4me3) or chromatin remodelers

  • Applications: Determine co-occupancy of c-Jun with specific epigenetic marks

  • Controls: Switch the order of antibodies to confirm results

• Integrative genomics approach:

  • Combine c-Jun ChIP-seq with:

    • ATAC-seq to assess chromatin accessibility

    • RNA-seq to correlate binding with gene expression

    • Hi-C to examine 3D chromatin architecture

  • Analysis: Identify c-Jun-dependent changes in chromatin organization

• Functional validation experiments:

  • CRISPR-Cas9 deletion of c-Jun binding sites

  • Site-directed mutagenesis of AP-1 motifs

  • c-Jun knockdown/knockout followed by ATAC-seq

  • Histone modification ChIP-qPCR at c-Jun target genes before and after c-Jun depletion

This comprehensive approach can reveal how c-Jun contributes to chromatin reorganization and epigenetic modifications, particularly at enhancers and super-enhancers regulating cell type-specific gene expression programs.

What are the potential applications of c-Jun(Ab-239) Antibody in single-cell protein analysis technologies?

c-Jun(Ab-239) Antibody can be adapted for emerging single-cell protein analysis techniques:

• Mass cytometry (CyTOF) integration:

  • Metal conjugation: Conjugate antibody with rare earth metals (e.g., lanthanides)

  • Panel design: Include in panels with 30+ other proteins

  • Sample preparation: Optimize nuclear permeabilization for transcription factor detection

  • Applications: Profile c-Jun activation in rare cell populations

  • Data analysis: Apply dimension reduction algorithms (t-SNE, UMAP) to visualize cellular heterogeneity

• Single-cell Western blotting:

  • Microfluidic adaptation: Optimize antibody concentration for reduced volumes

  • Detection system: Fluorophore-conjugated secondary antibodies

  • Quantification: Correlate c-Jun levels with other signaling proteins at single-cell resolution

  • Applications: Analyze signaling heterogeneity in cancer cells

• Proximity extension assay (PEA):

  • Approach: Split the antibody recognition and DNA barcode reporting functions

  • Application: Multiplex c-Jun detection with 90+ other proteins

  • Advantage: Requires minimal sample input (1 μL)

  • Limitation: Requires antibody pair with non-overlapping epitopes

• Microfluidic antibody capture:

  • Technology: Capture cells in nanoliter droplets with barcoded antibodies

  • Application: Correlate c-Jun protein levels with transcriptome

  • Integration: Combine with single-cell RNA-seq for multi-modal analysis

  • Expected insight: Connect c-Jun protein levels to downstream transcriptional programs

These emerging technologies enable unprecedented insights into c-Jun's role in cellular heterogeneity and its context-dependent functions in complex tissues.

How might c-Jun(Ab-239) Antibody be utilized in antibody-based therapeutic research applications?

While primarily a research tool, c-Jun(Ab-239) Antibody can inform therapeutic antibody development strategies:

• Antibody-drug conjugate (ADC) proof-of-concept studies:

  • Target validation: Use c-Jun(Ab-239) Antibody to validate c-Jun as a tumor-specific marker

  • Internalization studies: Track antibody internalization in c-Jun overexpressing cells

  • Conjugation strategies: Test various linker chemistries including disulfide re-bridging conjugation

  • Analysis framework: Compare with current ADC development platforms

• Intrabody development:

  • Antibody engineering: Express single-chain variable fragments (scFvs) based on c-Jun(Ab-239) binding domains

  • Applications: Develop c-Jun inhibitory intrabodies for cancer therapy research

  • Delivery systems: Viral vectors or nanoparticle-based delivery

  • Validation: Functional assays to confirm c-Jun inhibition

• Proteolysis-targeting chimera (PROTAC) research:

  • Approach: Use the targeting moiety from c-Jun(Ab-239) linked to E3 ligase ligands

  • Mechanism: Induce selective degradation of c-Jun protein

  • Applications: Study consequences of acute c-Jun depletion

  • Advantage: More selective than genetic knockout approaches

• Bispecific antibody concepts:

  • Design: Create bispecific constructs targeting c-Jun and effector cells

  • Applications: Explore targeted elimination of cells with nuclear c-Jun overexpression

  • Limitations: Challenges in targeting intracellular antigens

  • Alternative approach: Target surface proteins co-expressed with c-Jun

These applications leverage antibody engineering principles to explore novel therapeutic strategies targeting transcription factor activity in disease contexts .

What methodological considerations should researchers account for when using c-Jun(Ab-239) Antibody in spatial transcriptomics and multi-omics integration?

Integrating c-Jun(Ab-239) Antibody into spatial and multi-omics experiments requires specific methodological considerations:

• Spatial proteomics integration:

  • Compatible technologies: Imaging Mass Cytometry (IMC), Multiplexed Ion Beam Imaging (MIBI), Co-Detection by Indexing (CODEX)

  • Antibody validation: Confirm specificity in tissue sections with appropriate controls

  • Panel design: Include cell type markers, other signaling proteins, and microenvironment markers

  • Metal conjugation: Label antibody with lanthanide metals for IMC/MIBI

  • Analysis framework: Apply neighborhood analysis to identify spatial relationships

• Multi-modal single-cell analysis:

  • CITE-seq adaptation: Surface protein + transcriptome (though limited for nuclear targets)

  • Cellular indexing: Fixed cell barcoding with antibody detection

  • Sample preparation: Nuclear permeabilization optimization

  • Validation: Correlate protein detection with mRNA expression of target genes

  • Statistical approach: Apply multi-modal data integration algorithms

• Spatial transcriptomics integration:

  • Sequential workflow: Immunofluorescence with c-Jun(Ab-239) Antibody followed by spatial transcriptomics

  • Registration methods: Computational alignment of protein and transcriptome data

  • Resolution matching: Account for different resolution scales between methods

  • Analysis approach: Correlate c-Jun protein localization with spatially resolved gene expression

• Experimental design considerations:

  • Tissue preparation: Optimize fixation to preserve both protein epitopes and RNA integrity

  • Serial sections: Use adjacent sections for protein and RNA detection if simultaneous detection is challenging

  • Quality controls: Include spike-in controls and reference landmarks

  • Computational integration: Develop or apply algorithms that bridge protein and transcriptome data

These methodological considerations enable researchers to place c-Jun activity in its spatial and multi-omic context, revealing its role in tissue organization and cell-cell communication networks.