E2F3 Antibody

What is E2F3 and what is its significance in cellular research?

E2F3 is a transcription factor that belongs to the E2F family and plays a crucial role in regulating the cell cycle, particularly in the G1 to S phase transition where DNA synthesis occurs. The human E2F3 protein is approximately 49.2 kilodaltons in mass and exists in multiple isoforms, including isoform 2 which is 334 amino acids (37 kDa) . E2F3 functions as a transcription factor that activates genes necessary for cell cycle progression, making it essential for proper cell proliferation and differentiation .

Its activity is tightly regulated by interactions with retinoblastoma (Rb) protein, which when functional, binds to E2F3 and inhibits its transcriptional activity, thereby preventing premature entry into S phase . Dysregulation of E2F3 can lead to uncontrolled cell growth and is frequently implicated in cancer development, making it a significant target for cancer research . Understanding E2F3 dynamics is essential for developing therapeutic strategies targeting cell cycle dysregulation in various cancers.

How do I select the appropriate E2F3 antibody for my specific experimental needs?

Selecting the appropriate E2F3 antibody requires careful consideration of several factors:

• Application compatibility: First, determine which applications you need the antibody for (Western blot, immunoprecipitation, immunohistochemistry, etc.) and select antibodies validated for those specific applications. For example, the PG30 monoclonal antibody has been validated for western blotting (WB) and immunoprecipitation (IP) , while other antibodies may be specifically validated for immunocytochemistry (ICC) or immunofluorescence (IF) .

• Species reactivity: Ensure the antibody recognizes E2F3 in your experimental model organism. Available antibodies show reactivity with human, mouse, rat, cow, dog, guinea pig, horse, and rabbit E2F3, with varying degrees of cross-reactivity .

• Antibody type: Consider whether a monoclonal or polyclonal antibody is more suitable for your research. Monoclonal antibodies like PG30 (mouse IgG2a kappa) offer high specificity, while polyclonal antibodies may provide better sensitivity in certain applications .

• Epitope recognition: Some antibodies target specific regions of E2F3, such as C-terminal regions. The ABIN2780388 antibody, for example, targets the C-terminal region of human E2F3 . This is particularly important if you're interested in detecting specific isoforms or domains.

• Conjugation needs: Determine whether you need a conjugated antibody (e.g., HRP, FITC, PE) or an unconjugated form depending on your detection method .

What are the most common applications for E2F3 antibodies in research?

E2F3 antibodies are utilized in a variety of research applications, each providing unique insights into E2F3 function and expression:

• Western Blotting (WB): The most common application, used to detect and quantify E2F3 protein levels in cell or tissue lysates. Multiple antibodies including PG30 and ABIN2780388 are validated for WB applications .

• Immunoprecipitation (IP): Used to isolate E2F3 and its binding partners from complex protein mixtures. The PG30 antibody has been validated for this application .

• Immunocytochemistry (ICC) and Immunofluorescence (IF): Used to visualize the subcellular localization of E2F3 in fixed cells, revealing its nuclear distribution patterns during different cell cycle phases .

• Immunohistochemistry (IHC): Applied to tissue sections to examine E2F3 expression patterns in different cell types within tissues, particularly useful in cancer research .

• ELISA (Enzyme-Linked Immunosorbent Assay): Used for quantitative detection of E2F3 in samples .

• Proximity Ligation Assay (PLA): Advanced application used to detect protein-protein interactions involving E2F3, such as its association with Rb protein .

• Chromatin Immunoprecipitation (ChIP): Used to identify genomic regions bound by E2F3, helping to identify its target genes.

How can I validate the specificity of an E2F3 antibody for my experiments?

Validating antibody specificity is crucial for generating reliable data. For E2F3 antibodies, consider implementing these validation strategies:

• Positive and negative controls: Use cell lines or tissues known to express high levels of E2F3 (positive control) and those with low or no expression (negative control). Cancer cell lines often express high levels of E2F3 and make good positive controls.

• Knockout/knockdown validation: Validate specificity by comparing antibody detection in wild-type samples versus those where E2F3 has been knocked down (siRNA/shRNA) or knocked out (CRISPR-Cas9). A specific antibody should show reduced or absent signal in knockdown/knockout samples.

• Blocking peptide experiments: Pre-incubate the antibody with the immunizing peptide before application to your sample. If the antibody is specific, the peptide should block binding and eliminate signal. Many suppliers, including those offering ABIN2780388, provide information about the immunizing peptide used .

• Molecular weight verification: Confirm that the detected band in Western blot corresponds to the expected molecular weight of E2F3 (approximately 49.2 kDa for full-length protein, or 37 kDa for isoform 2) .

• Multiple antibody concordance: Use multiple antibodies targeting different epitopes of E2F3 to confirm results. Consistent findings across different antibodies increase confidence in specificity.

• Isoform analysis: If studying specific E2F3 isoforms, verify that the antibody can distinguish between them. Some antibodies are isoform-specific, such as those that can differentiate E2F3a from E2F3b.

How can I distinguish between E2F3a and E2F3b isoforms in my experiments?

Distinguishing between E2F3a and E2F3b isoforms requires careful antibody selection and experimental design:

• Isoform-specific antibodies: Select antibodies that specifically recognize unique epitopes in either E2F3a or E2F3b. E2F3a is the full-length protein (approximately 49.2 kDa), while E2F3b is a shorter isoform (approximately 37 kDa) that lacks the N-terminal domain present in E2F3a .

• Western blot resolution: Use gradient gels (e.g., 7.5-12%) with extended running times to achieve clear separation between the 49.2 kDa E2F3a and 37 kDa E2F3b bands. Note that some antibodies, like those targeting the C-terminal region, can detect both isoforms but will show different molecular weight bands .

• Immunoprecipitation followed by mass spectrometry: For definitive isoform identification, consider immunoprecipitating E2F3 proteins using a pan-E2F3 antibody followed by mass spectrometry analysis to identify isoform-specific peptides.

• RT-PCR analysis in parallel: Complement protein-level detection with RT-PCR using isoform-specific primers to confirm the expression of E2F3a versus E2F3b transcripts.

• Functional assays: E2F3a and E2F3b have different functional properties; E2F3a is generally considered an activator while E2F3b can function as a repressor in certain contexts. Design functional reporter assays to differentiate their activities.

What are the optimal protocols for studying E2F3 interactions with Rb protein using antibody-based approaches?

Studying E2F3-Rb interactions is crucial for understanding cell cycle regulation. Here are optimal protocols using antibody-based approaches:

• Co-immunoprecipitation (Co-IP):

  • Lyse cells in non-denaturing buffer containing phosphatase inhibitors (critical as Rb-E2F3 interactions are phosphorylation-dependent)

  • Immunoprecipitate with E2F3 antibody (e.g., PG30) or Rb antibody

  • Analyze precipitated complexes by Western blot using antibodies against the reciprocal protein

  • Include controls for non-specific binding (IgG control) and input samples

• Proximity Ligation Assay (PLA):

  • Fix cells at specific cell cycle stages (serum starvation followed by release)

  • Incubate with primary antibodies against E2F3 and Rb

  • Apply PLA probes and perform ligation and amplification

  • Visualize interaction foci using fluorescence microscopy

  • Quantify interaction events per cell using image analysis software

• Chromatin Immunoprecipitation (ChIP):

  • Perform sequential ChIP (first with E2F3 antibody, then with Rb antibody) to identify genomic regions bound by E2F3-Rb complexes

  • Use ABIN2780388 or similar antibodies validated for ChIP applications

  • Include cell cycle synchronization to capture phase-specific interactions

• FRET-based assays:

  • Use fluorescently labeled antibodies against E2F3 and Rb

  • Measure FRET signals as indicators of protein proximity

  • Analyze cell cycle-dependent changes in FRET efficiency

• In vitro binding assays:

  • Use recombinant E2F3 proteins and immobilized antibodies to create affinity columns

  • Pass Rb-containing lysates through the column and analyze binding under different phosphorylation conditions

What are the common troubleshooting strategies for E2F3 antibody applications in challenging samples?

Working with E2F3 antibodies in challenging samples can present various technical difficulties. Here are effective troubleshooting strategies:

• Weak or no signal in Western blot:

  • Increase antibody concentration or incubation time

  • Optimize protein extraction protocol to ensure E2F3 is efficiently solubilized

  • Try different blocking agents (BSA vs. milk) as E2F3 antibodies may be sensitive to blocking conditions

  • Consider using conjugated antibodies or signal amplification systems

  • Use fresh samples as E2F3 may degrade during storage

• High background in immunostaining:

  • Increase blocking time and concentration

  • Optimize antibody dilution (generally 1:100 to 1:500 for most E2F3 antibodies)

  • Include additional washing steps with higher detergent concentration

  • Use monoclonal antibodies like PG30 which may provide cleaner staining

  • Consider using fluorophore-conjugated secondary antibodies for better signal-to-noise ratio

• Inconsistent results across experiments:

  • Standardize cell culture conditions, as E2F3 expression varies throughout the cell cycle

  • Use synchronized cell populations when possible

  • Implement internal loading controls and normalization strategies

  • Consider batch effects in antibody performance and use the same lot where possible

• Problems with formalin-fixed tissues:

  • Optimize antigen retrieval methods (citrate buffer at pH 6.0 often works well)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use antibodies specifically validated for IHC applications on FFPE tissues

  • Consider using amplification systems for enhanced sensitivity

• Cross-reactivity issues:

  • Use antibodies with confirmed specificity for your species of interest

  • Validate using genetic approaches (siRNA, CRISPR knockout)

  • Consider using multiple antibodies targeting different epitopes to confirm findings

How can I effectively use E2F3 antibodies in cancer research and what are the key considerations?

E2F3 antibodies are valuable tools in cancer research due to E2F3's frequent dysregulation in malignancies. Here are effective strategies and considerations:

• Tumor tissue analysis:

  • Use immunohistochemistry with E2F3 antibodies to assess expression levels and subcellular localization in tumor versus normal tissues

  • Consider double staining with proliferation markers (Ki-67) to correlate E2F3 expression with proliferative index

  • Optimize antibody concentration for each tumor type as expression levels vary significantly across cancer types

  • Use antibodies validated specifically for IHC in human tissues

• Prognostic biomarker development:

  • Establish standardized scoring systems for E2F3 immunostaining intensity and distribution

  • Correlate E2F3 expression patterns with clinical outcomes using tissue microarrays

  • Consider isoform-specific antibodies as E2F3a and E2F3b may have different prognostic implications

  • Implement rigorous statistical analysis to validate prognostic value

• Therapeutic target identification:

  • Use ChIP-seq with E2F3 antibodies to identify cancer-specific E2F3 target genes

  • Apply E2F3 antibodies in drug screening assays to identify compounds that modulate E2F3 activity

  • Monitor E2F3 levels using Western blot to assess response to cell cycle-targeted therapies

• Cell cycle checkpoint analysis:

  • Synchronize cells and collect at different time points to analyze E2F3 dynamics during cell cycle

  • Use phospho-specific antibodies to distinguish active versus inactive E2F3 forms

  • Combine with flow cytometry to correlate E2F3 status with cell cycle phase

• Resistance mechanism studies:

  • Compare E2F3 levels and localization in sensitive versus resistant cancer cells

  • Analyze E2F3-Rb interaction status in therapy-resistant models using co-IP approaches with PG30 or similar antibodies

  • Assess E2F3 target gene expression in response to therapy to identify altered regulatory networks

What are the optimal storage and handling conditions for maintaining E2F3 antibody efficacy?

Proper storage and handling of E2F3 antibodies is crucial for maintaining their performance over time:

• Storage temperature:

  • Store most unconjugated E2F3 antibodies at -20°C for long-term storage

  • Store working aliquots at 4°C for up to one month

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

  • Conjugated antibodies (FITC, PE, HRP) may require special storage considerations; follow manufacturer guidelines

• Buffer composition:

  • Most E2F3 antibodies are supplied in buffer containing PBS, glycerol, and carrier proteins

  • Some formulations include preservatives like sodium azide

  • Do not dilute stock antibody unless preparing working aliquots

• Working dilutions:

  • For Western blot: Typically 1:500 to 1:2000 dilution

  • For IHC/ICC: Typically 1:100 to 1:500 dilution

  • Always optimize dilutions for each application and sample type

  • Prepare fresh working dilutions for each experiment

• Shipping and temporary storage:

  • Most E2F3 antibodies are shipped with ice packs

  • Upon receipt, immediately transfer to recommended storage conditions

  • Brief exposure to room temperature during handling is generally acceptable

• Stability considerations:

  • Typical shelf life is 12-24 months when properly stored

  • Avoid contamination by using sterile technique when handling

  • Monitor performance over time; decreased activity may indicate degradation

How can I optimize E2F3 antibody-based Western blot protocols for maximum sensitivity and specificity?

Optimizing Western blot protocols for E2F3 detection requires attention to several key parameters:

• Sample preparation:

  • Use RIPA or NP-40 buffer with protease and phosphatase inhibitors

  • Include DNase treatment to reduce viscosity from nuclear material

  • Heat samples at 95°C for 5 minutes in Laemmli buffer with reducing agent

  • Load 20-50 μg of total protein per lane for cell lysates

• Gel selection and transfer:

  • Use 10% SDS-PAGE gels for optimal resolution of E2F3 (49.2 kDa) and its isoforms

  • Transfer to PVDF membranes (preferred over nitrocellulose for nuclear proteins)

  • Use semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour

• Blocking and antibody incubation:

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

  • For phospho-specific detection, use 5% BSA instead of milk

  • Incubate with primary E2F3 antibody (e.g., PG30, ABIN2780388) at 1:1000 dilution overnight at 4°C

  • Wash extensively (4 × 5 minutes) with TBST before and after secondary antibody

• Detection optimization:

  • Use HRP-conjugated secondary antibodies or consider directly conjugated E2F3 antibodies

  • For enhanced sensitivity, use chemiluminescent substrates with signal enhancers

  • Optimize exposure time to avoid saturation while capturing weak signals

  • Consider using fluorescently labeled secondary antibodies for multiplex detection and quantification

• Controls and troubleshooting:

  • Always include positive control lysate from cells known to express E2F3

  • Use recombinant E2F3 protein as a standard for size verification

  • Include loading controls (β-actin, GAPDH) but note that these may not be ideal for nuclear proteins

  • Consider using nuclear-specific loading controls like Lamin B1

What methodologies are recommended for studying E2F3 post-translational modifications using specific antibodies?

Studying E2F3 post-translational modifications (PTMs) requires specialized approaches:

• Phosphorylation analysis:

  • Use phospho-specific E2F3 antibodies when available

  • Treat samples with lambda phosphatase as a negative control

  • Implement Phos-tag SDS-PAGE to enhance separation of phosphorylated forms

  • Use kinase inhibitors or activators to manipulate E2F3 phosphorylation status

  • Perform parallel immunoprecipitation with general E2F3 antibodies followed by phospho-specific Western blotting

• Ubiquitination detection:

  • Add proteasome inhibitors (MG132) to cell culture prior to lysis

  • Perform immunoprecipitation with E2F3 antibodies like PG30

  • Blot for ubiquitin to detect ubiquitinated E2F3 forms

  • Use denaturing conditions during lysis to disrupt protein-protein interactions

  • Consider tandem ubiquitin binding entity (TUBE) pulldown followed by E2F3 detection

• Acetylation analysis:

  • Treat cells with histone deacetylase inhibitors to enhance acetylation

  • Immunoprecipitate E2F3 and probe with pan-acetyl-lysine antibodies

  • Use mass spectrometry to identify specific acetylated residues

  • Compare acetylation status across cell cycle phases

• SUMOylation detection:

  • Express tagged SUMO proteins in cells

  • Immunoprecipitate with E2F3 antibodies under denaturing conditions

  • Blot for SUMO tags to identify SUMOylated E2F3

  • Use SUMO-specific proteases as negative controls

• Integrated approaches:

  • Combine immunoprecipitation with mass spectrometry for comprehensive PTM identification

  • Create PTM-specific mutants to validate antibody specificity

  • Use proximity ligation assays to detect E2F3 interactions with PTM-adding enzymes in situ

  • Develop cell-free systems with purified components to study PTM mechanisms

How do monoclonal versus polyclonal E2F3 antibodies compare in different research applications?

Choosing between monoclonal and polyclonal E2F3 antibodies depends on your specific research requirements:

Selection criteria should include:

• Application specificity: For quantitative Western blots, monoclonals like PG30 provide consistent results . For detecting E2F3 in fixed tissues, polyclonals may offer better sensitivity.

• Epitope accessibility: Consider whether your experimental conditions might mask particular epitopes. Polyclonals recognize multiple epitopes and may be more robust across different sample preparation methods.

• Signal strength requirements: If detecting low abundance E2F3 (as in some normal tissues), polyclonals may provide better sensitivity. For highly expressed E2F3 (as in many cancer cells), monoclonals offer cleaner results.

• Reproducibility needs: For longitudinal studies requiring consistent results over time, monoclonal antibodies like PG30 offer superior batch-to-batch consistency .

What are the considerations for choosing between different conjugated forms of E2F3 antibodies?

Selecting the appropriate conjugated form of E2F3 antibody is critical for experimental success:

Selection considerations: