JUN (Ab-170) Antibody

What exactly is the JUN (Ab-170) Antibody and what is its target specificity?

The JUN (Ab-170) Antibody is a rabbit polyclonal antibody specifically designed to detect endogenous levels of total c-Jun protein . The antibody recognizes an epitope corresponding to amino acids 168-172 (P-V-Y-A-N) of human c-Jun . c-Jun functions as a transcription factor that recognizes and binds to the enhancer heptamer motif 5′-TGA[CG]TCA-3′ and is a critical component of the AP-1 (Activator Protein 1) transcription factor complex .

Technical specifications:

• Host/Species: Rabbit

• Clonality: Polyclonal

• Immunogen: Peptide sequence around aa. 168~172 (P-V-Y-A-N) derived from Human c-Jun

• Target aliases: AH119; AP1; Jun A; c-Jun; p39

• UniProt ID: P05412

What species cross-reactivity has been validated for the JUN (Ab-170) Antibody?

The JUN (Ab-170) Antibody has been experimentally validated to detect c-Jun in human, mouse, and rat samples . This cross-species reactivity is due to the high conservation of the epitope region (amino acids 168-172) across these mammalian species . When designing experiments with other species, validation studies should be performed as cross-reactivity with non-validated species cannot be guaranteed.

What applications has the JUN (Ab-170) Antibody been validated for?

The JUN (Ab-170) Antibody has been validated for the following applications:

Example validation data includes Western blot analysis of Hela cells and immunohistochemical staining of human breast carcinoma tissue .

How does the epitope specificity of JUN (Ab-170) Antibody affect detection of c-Jun in different cellular contexts?

For optimal results in complex cellular environments:

• Use appropriate sample preparation methods to maintain protein conformation

• Consider complementary approaches (e.g., using another c-Jun antibody targeting a different epitope) for validation

• Evaluate potential masking effects in your experimental system

Can the JUN (Ab-170) Antibody distinguish between different phosphorylated states of c-Jun?

The JUN (Ab-170) Antibody detects total c-Jun protein regardless of its phosphorylation status . It targets amino acids 168-172, while the primary phosphorylation sites of c-Jun are at serine residues 63, 73 and threonines 91, 93, and 239 . For research requiring distinction between phosphorylated and non-phosphorylated c-Jun, phospho-specific antibodies should be used alongside this total c-Jun antibody.

For researchers investigating c-Jun phosphorylation states, specialized phospho-specific antibodies are available for various sites including:

• c-Jun (phospho Ser63)

• c-Jun (phospho Thr91)

• c-Jun (phospho Thr93)

• c-Jun (phospho Thr239)

• c-Jun (phospho Ser243)

What is known about JUN (Ab-170) Antibody's performance in cancer research applications?

The JUN (Ab-170) Antibody has demonstrated utility in cancer research applications, particularly in detecting c-Jun expression changes in various tumor types . Validation data shows successful immunohistochemical staining of human breast carcinoma tissue . c-Jun is frequently overexpressed or hyperactivated in many cancer types due to its role in controlling cell proliferation, survival, and apoptosis.

Researchers using this antibody in cancer studies should consider:

• Optimizing antigen retrieval methods for formalin-fixed tissues

• Including appropriate positive and negative controls

• Using quantitative analysis methods to accurately measure expression levels

• Correlating c-Jun detection with other cancer biomarkers for comprehensive analysis

What are the optimal protocols for using JUN (Ab-170) Antibody in Western blot applications?

For optimal Western blot results with JUN (Ab-170) Antibody, the following protocol is recommended:

Sample preparation:

• Use standard cell/tissue lysis buffers containing protease inhibitors

• Denature samples in SDS loading buffer at 95°C for 5 minutes

• Load 20-50 μg of total protein per lane

Western blot protocol:

• Separate proteins on 10-12% SDS-PAGE gel (optimal for 43 kDa c-Jun)

• Transfer to PVDF or nitrocellulose membrane

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

• Incubate with JUN (Ab-170) Antibody at 1:500-1:1000 dilution overnight at 4°C

• Wash 3-5 times with TBST

• Incubate with appropriate HRP-conjugated secondary anti-rabbit antibody

• Develop using ECL detection system

Expected result: A primary band at approximately 43 kDa corresponding to c-Jun .

What are the recommended protocols for immunohistochemistry using JUN (Ab-170) Antibody?

For optimal immunohistochemistry results with JUN (Ab-170) Antibody:

Protocol for FFPE sections:

• De-paraffinize and rehydrate sections

• Perform heat-mediated antigen retrieval (recommended: citrate buffer pH 6.0)

• Block endogenous peroxidase with 3% H₂O₂

• Block non-specific binding with 5-10% normal serum

• Incubate with JUN (Ab-170) Antibody at 1:50-1:100 dilution overnight at 4°C

• Wash with PBS or TBS

• Apply appropriate detection system (e.g., HRP-polymer or biotin-streptavidin)

• Develop with DAB or other appropriate chromogen

• Counterstain, dehydrate, and mount

Expected results: Nuclear staining in c-Jun expressing cells, with potential cytoplasmic staining depending on cellular status and fixation methods .

How should researchers validate JUN (Ab-170) Antibody specificity in their experimental systems?

Validation of antibody specificity is crucial for reliable research results. For JUN (Ab-170) Antibody, consider these validation approaches:

• Positive and negative controls:

  • Positive: Cell lines known to express c-Jun (e.g., HeLa cells)

  • Negative: c-Jun knockout or knockdown samples

• Blocking peptide competition:

  • Pre-incubate antibody with excess immunizing peptide (P-V-Y-A-N sequence)

  • Compare results with and without blocking peptide

• Molecular weight verification:

  • Confirm detection of a band at the expected molecular weight (~43 kDa)

• Multiple antibody validation:

  • Compare results with another c-Jun antibody targeting a different epitope

• Correlation with mRNA expression:

  • Compare protein detection with c-Jun mRNA levels by RT-PCR or RNA-seq

What are common causes of weak or absent signal when using JUN (Ab-170) Antibody?

When experiencing weak or absent signal with JUN (Ab-170) Antibody, consider these potential issues and solutions:

How can researchers address non-specific bands in Western blot using JUN (Ab-170) Antibody?

Non-specific bands are a common challenge in Western blotting. For JUN (Ab-170) Antibody, try these approaches:

• Optimize blocking:

  • Test different blocking agents (BSA vs. milk)

  • Increase blocking time or concentration

• Adjust antibody concentration:

  • Test a dilution series (e.g., 1:250, 1:500, 1:1000, 1:2000)

  • Find optimal balance between specific signal and background

• Increase washing stringency:

  • Add additional wash steps

  • Increase salt concentration in wash buffer

  • Add low concentrations of detergent (0.1-0.3% Tween-20)

• Use gradient gels:

  • Improve separation of proteins in the molecular weight range of interest

• Validate with controls:

  • Compare with lysates from c-Jun knockdown or knockout samples

  • Use blocking peptide competition to identify specific bands

What factors affect the reproducibility of results with JUN (Ab-170) Antibody?

Ensuring reproducible results with JUN (Ab-170) Antibody requires attention to several factors:

• Antibody handling and storage:

  • Aliquot stock solution to avoid freeze-thaw cycles

  • Store at recommended temperature (-20°C long-term; 4°C short-term)

  • Check expiration date (typically 12 months from receipt)

• Sample preparation consistency:

  • Standardize lysis buffers and extraction methods

  • Use fresh protease/phosphatase inhibitors

  • Maintain consistent protein concentrations between experiments

• Experimental conditions:

  • Document and maintain consistent antibody dilutions

  • Standardize incubation times and temperatures

  • Use the same detection systems across experiments

• Quantification methods:

  • Use appropriate loading controls

  • Apply consistent image acquisition parameters

  • Employ standardized quantification methods

By systematically controlling these variables, researchers can significantly improve the reproducibility of their c-Jun detection results.

How has JUN (Ab-170) Antibody been utilized in cancer research studies?

The JUN (Ab-170) Antibody has been employed in various cancer research applications:

• Expression profiling:

  • Detection of c-Jun levels in different cancer types

  • Correlation of c-Jun expression with clinical parameters

• Signaling pathway analysis:

  • Investigation of MAPK/JNK pathway activation leading to c-Jun upregulation

  • Analysis of c-Jun's role in AP-1 complex formation in tumor cells

• Diagnostic applications:

  • Evaluation of c-Jun as a potential biomarker in breast carcinoma

  • Assessment of nuclear localization patterns in malignant tissues

Researchers have successfully used this antibody for immunohistochemical staining of human breast carcinoma tissue, demonstrating its utility in analyzing c-Jun expression in clinical samples .

What insights can studying c-Jun with JUN (Ab-170) Antibody provide about cellular stress responses?

c-Jun is a central mediator of cellular stress responses, and studying it with JUN (Ab-170) Antibody can reveal:

• Stress-induced expression changes:

  • Quantification of c-Jun upregulation following UV radiation, oxidative stress, inflammatory cytokines, and other stressors

  • Temporal dynamics of c-Jun expression during stress response and recovery

• Subcellular localization:

  • Nuclear accumulation of c-Jun following stress activation

  • Association with specific nuclear structures or chromatin regions

• Context-dependent function:

  • c-Jun's dual role in promoting both cell survival and apoptosis depending on cellular context and stimulus type

  • Integration of multiple stress response pathways through c-Jun regulation

Methodological considerations for stress response studies include careful timing of sample collection, appropriate stress induction protocols, and correlation with functional outcomes like cell viability or gene expression changes.