JUN (Ab-63) Antibody

What is the c-Jun protein and what cellular functions does it regulate?

c-Jun is a protein encoded by the JUN gene, which is the putative transforming gene of avian sarcoma virus 17. It functions as a critical component of the AP-1 (Activator Protein-1) transcription factor complex that regulates gene expression by interacting directly with specific DNA sequences . The gene is intronless and mapped to chromosome 1p32-p31, a region frequently involved in both translocations and deletions in human malignancies .

At the molecular level, c-Jun participates in numerous cellular processes including:

• Cell proliferation and differentiation

• Apoptosis regulation

• Stress response pathways

• Inflammatory signaling

• Oncogenic transformation

The protein's activity is primarily regulated through phosphorylation events at specific serine residues, particularly serine 63 and 73, which enhance its transcriptional activity in response to various stimuli.

What epitope does JUN (Ab-63) Antibody specifically recognize?

JUN (Ab-63) Antibody specifically recognizes the peptide sequence around amino acids 61-65 (L-T-S-P-D) derived from Human c-Jun . This region is particularly significant as it contains serine 63, a critical phosphorylation site that regulates c-Jun transcriptional activity. The antibody detects endogenous levels of total c-Jun protein, regardless of phosphorylation status at this site .

The antibody was generated by immunizing rabbits with a synthetic peptide corresponding to this region conjugated to KLH (Keyhole Limpet Hemocyanin) carrier protein. It was subsequently purified through affinity chromatography using epitope-specific peptide columns to ensure high specificity .

What applications has JUN (Ab-63) Antibody been validated for?

JUN (Ab-63) Antibody has been validated for multiple experimental applications:

Validation data typically includes experiments with positive control samples such as human HeLa, K562, Jurkat, and U-87MG cell lysates for Western blot applications, and human renal cell carcinoma tissue for IHC applications .

What species reactivity has been confirmed for JUN (Ab-63) Antibody?

The JUN (Ab-63) Antibody has been confirmed to react with c-Jun protein from multiple species:

• Human (primary reactivity)

• Mouse

• Rat

Cross-reactivity testing ensures that the antibody recognizes the conserved epitope region (L-T-S-P-D) across these species. This cross-species reactivity is particularly valuable for comparative studies examining c-Jun expression and function across different model organisms .

What are the optimal storage and handling conditions for JUN (Ab-63) Antibody?

For optimal performance and longevity, JUN (Ab-63) Antibody should be stored and handled according to these guidelines:

• Long-term storage: -20°C (recommended)

• Short-term use: 4°C

• Formulation: Supplied at 1.0mg/mL in phosphate buffered saline (without Mg2+ and Ca2+), pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol

• Avoid: Repeated freeze/thaw cycles that can degrade antibody quality

• Reconstitution: For lyophilized format, reconstitute with 0.2 ml distilled water to yield a concentration of 500 μg/ml

Proper storage is critical for maintaining antibody performance in experimental applications. Aliquoting the antibody before freezing can prevent degradation associated with freeze/thaw cycles.

How can researchers optimize Western blot protocols for JUN (Ab-63) Antibody?

For optimal Western blot results with JUN (Ab-63) Antibody, researchers should implement this methodological approach:

Sample Preparation:

• Use 30 μg of protein per lane for cell lysates under reducing conditions

• Include positive controls such as Hela, K562, Jurkat, or U-87MG cell lysates

Electrophoresis Conditions:

• Use 5-20% SDS-PAGE gel

• Run at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours

Transfer Parameters:

• Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

• Verify transfer efficiency with reversible protein stain before blocking

Blocking and Antibody Incubation:

• Block membrane with 5% non-fat milk in TBS for 1.5 hours at room temperature

• Incubate with JUN (Ab-63) Antibody at 0.5 μg/mL (1:500-1:1000 dilution) overnight at 4°C

• Wash with TBS-0.1% Tween 3 times, 5 minutes each

• Probe with goat anti-rabbit IgG-HRP secondary antibody at 1:5000 dilution for 1.5 hours at room temperature

Detection:

• Develop using enhanced chemiluminescence (ECL) detection system

• Expected band size for c-Jun is approximately 36-39 kDa

Troubleshooting Tips:

• If background is high, increase washing steps or decrease primary antibody concentration

• If signal is weak, extend exposure time or increase antibody concentration

What are the best practices for using JUN (Ab-63) Antibody in immunohistochemistry applications?

For effective immunohistochemical detection of c-Jun using JUN (Ab-63) Antibody, follow these methodological guidelines:

Tissue Preparation:

• Fix tissues in 10% neutral buffered formalin

• Embed in paraffin and section at 4-6 μm thickness

• Mount sections on positively charged slides

Antigen Retrieval:

• Perform heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

• For enzyme antigen retrieval, use standard enzyme antigen retrieval reagent for 15 minutes

Staining Protocol:

• Block endogenous peroxidase activity with hydrogen peroxide

• Block non-specific binding with 10% goat serum

• Incubate with JUN (Ab-63) Antibody at 2-5 μg/ml overnight at 4°C

• For detection, use appropriate secondary antibody system such as Peroxidase Conjugated Goat Anti-rabbit IgG (incubate for 30 minutes at 37°C)

• Develop with DAB chromogen and counterstain with hematoxylin

• Mount with permanent mounting medium

Positive Control Tissues:

• Human renal cell carcinoma tissue

• Human renal pelvis squamous metaplasia tissue

Expected Results:

• c-Jun primarily exhibits nuclear localization with occasionally cytoplasmic staining

• Expression patterns vary by tissue type and pathological state

How can JUN (Ab-63) Antibody be used to study phosphorylation-dependent signaling pathways?

While JUN (Ab-63) Antibody recognizes total c-Jun protein regardless of phosphorylation status, it can be utilized in combination with phospho-specific antibodies to study signaling pathways:

Experimental Approach:

• Parallel Immunoblotting: Run duplicate samples on separate gels, probing one with JUN (Ab-63) Antibody and the other with phospho-specific c-Jun (S63) antibody

• Quantitative Analysis:

  • Normalize phospho-c-Jun signal to total c-Jun detected by JUN (Ab-63) Antibody

  • Calculate phosphorylation ratios under different experimental conditions

• Pathway Stimulation Experiments:

  • Treat cells with pathway activators (e.g., TNF-α, UV radiation, growth factors)

  • Monitor changes in c-Jun phosphorylation relative to total protein levels

  • Combine with inhibitors to verify pathway specificity

• Antibody Array Approach:

  • Implement antibody array systems for insulin signaling pathways

  • Include JUN (Ab-63) Antibody alongside phospho-specific antibodies

  • Analyze pathway activation profiles across different experimental conditions

• Time-Course Studies:

  • Monitor both total and phosphorylated c-Jun levels over time following stimulus

  • Determine activation and deactivation kinetics of the signaling pathway

This methodology allows researchers to distinguish between changes in c-Jun expression versus changes in its phosphorylation state, providing insights into signaling dynamics.

How can researchers optimize immunofluorescence protocols with JUN (Ab-63) Antibody?

For high-quality immunofluorescence results with JUN (Ab-63) Antibody, implement this optimized protocol:

Cell Preparation:

• Culture cells on glass coverslips or chamber slides

• Fix using 4% paraformaldehyde and permeabilize with permeabilization buffer

Staining Protocol:

• Block with 10% normal goat serum to reduce non-specific binding

• Incubate with JUN (Ab-63) Antibody at 5 μg/mL dilution overnight at 4°C

• For co-staining experiments, combine with other primary antibodies (e.g., anti-Beta Tubulin)

• Use appropriate fluorescently conjugated secondary antibodies (e.g., DyLight®488 Conjugated Goat Anti-Rabbit IgG) at 1:100 dilution

• Incubate for 30 minutes at 37°C

• Counterstain nuclei with DAPI

• Mount with anti-fade mounting medium

Controls:

• Include negative controls (omitting primary antibody)

• Use A431 cells as positive control

• For validation, compare nuclear staining pattern with DAPI counterstain

Imaging Parameters:

• Visualize using appropriate filter sets for the fluorophores used

• Capture z-stack images to ensure complete signal detection

• Compare c-Jun localization with other cellular markers

Optimization Tips:

• Adjust antibody concentration based on signal-to-noise ratio

• Optimize permeabilization conditions for nuclear antigen access

• For weak signals, extend primary antibody incubation time

What considerations are important when using JUN (Ab-63) Antibody in flow cytometry applications?

For successful flow cytometry experiments with JUN (Ab-63) Antibody, researchers should follow these methodological guidelines:

Sample Preparation:

• Fix cells with 4% paraformaldehyde

• Permeabilize cells with appropriate permeabilization buffer to allow antibody access to intracellular antigens

• Block with 10% normal goat serum to minimize non-specific binding

Staining Protocol:

• Incubate with JUN (Ab-63) Antibody at 1-3 μg per 1×10^6 cells

• Use appropriate fluorochrome-conjugated secondary antibody

• Include proper compensation controls when performing multicolor analysis

Controls:

• Use U20S cells as positive control

• Include isotype control antibody (rabbit IgG) at equivalent concentration

• Perform unstained and secondary-only controls

Analysis Considerations:

• Gate on viable cell population

• Analyze intracellular c-Jun expression using appropriate statistical methods

• For multiparameter analysis, consider co-staining with markers of cell cycle or activation

Expected Results:

• c-Jun expression may vary across cell cycle stages

• Stimulus-induced changes in c-Jun expression can be quantitatively measured

• Population heterogeneity can be assessed through histogram analysis

How can JUN (Ab-63) Antibody be integrated into multiplexed antibody arrays for signaling pathway analysis?

Researchers can effectively incorporate JUN (Ab-63) Antibody into multiplexed antibody arrays using these methodological approaches:

Array Construction:

• Covalently immobilize JUN (Ab-63) Antibody alongside other signaling pathway antibodies on glass surfaces coated with polymeric 3D material

• Include both total protein and phospho-specific antibodies for comprehensive pathway analysis

Sample Processing:

• Label proteins in cell lysates with biotin

• Capture targeted proteins with immobilized antibodies on the array

• Detect with Cy3-labeled streptavidin or other fluorescent detection systems

Analytical Approaches:

• Compare relative signal intensities across different experimental conditions

• Normalize target protein signals to appropriate housekeeping proteins

• Perform cluster analysis to identify coordinated signaling responses

Validation Methods:

• Confirm key findings using conventional Western blot

• Validate with multiplexed bead array assays for orthogonal confirmation

• Perform follow-up functional studies on identified pathway components

Application Areas:

• Analyze insulin signaling pathway defects

• Investigate c-Jun's role in stress-activated protein kinase cascades

• Study oncogenic signaling networks in cancer models

This approach enables comprehensive analysis of c-Jun's involvement in complex signaling networks and provides a versatile method for detecting both total protein levels and post-translational modifications in normal and pathological states .