EIF4EBP1 (Ab-64) Antibody

What is EIF4EBP1 and what is its biological function?

EIF4EBP1 (also known as 4E-BP1, PHAS-I) is a translation repressor protein that regulates eIF4E activity by preventing its assembly into the eIF4F complex. In its hypophosphorylated form, EIF4EBP1 competes with EIF4G1/EIF4G3 and strongly binds to EIF4E, leading to repression of translation. Conversely, when hyperphosphorylated, EIF4EBP1 dissociates from EIF4E, allowing interaction between EIF4G1/EIF4G3 and EIF4E and enabling translation initiation .

EIF4EBP1 mediates the regulation of protein translation in response to various stimuli including hormones, growth factors, and other signals that act through the MAP kinase and mTORC1 pathways . It plays critical roles in cell growth, proliferation, and various disease states, particularly cancer.

What is the EIF4EBP1 (Ab-64) Antibody specifically designed to detect?

The EIF4EBP1 (Ab-64) Antibody is a rabbit polyclonal antibody that specifically detects endogenous levels of total 4E-BP1 protein. It was generated using a synthesized non-phosphopeptide derived from human 4E-BP1 around the phosphorylation site of serine 65 (R-N-S(p)-P-V) . This antibody recognizes both phosphorylated and non-phosphorylated forms of the protein and has been validated for detecting EIF4EBP1 with a molecular weight of approximately 15 kDa in SDS-PAGE applications .

What applications is the EIF4EBP1 (Ab-64) Antibody suitable for?

The EIF4EBP1 (Ab-64) Antibody has been validated for multiple applications:

• Western Blotting (WB): Recommended dilution range of 1:500 to 1:3000

• Immunohistochemistry (IHC): Recommended dilution range of 1:50 to 1:100

• ELISA: Used in various ELISA formats, including cell-based ELISA systems

The antibody has been tested and validated in these applications using human, mouse, and rat samples .

What is the recommended protocol for using EIF4EBP1 (Ab-64) Antibody in Western blotting?

For optimal Western blotting results with EIF4EBP1 (Ab-64) Antibody:

• Prepare cell/tissue lysates under reducing conditions.

• Separate proteins via SDS-PAGE (expect EIF4EBP1 at approximately 15 kDa).

• Transfer proteins to a membrane (PVDF or nitrocellulose).

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

• Incubate with primary EIF4EBP1 (Ab-64) Antibody at a dilution of 1:500 to 1:3000 in blocking buffer overnight at 4°C.

• Wash 3-5 times with TBST.

• Incubate with appropriate HRP-conjugated secondary antibody.

• Detect signal using enhanced chemiluminescence or infrared imaging systems .

A validation example shows detection of EIF4EBP1 in Jurkat cells treated with insulin (0.01U/ml, 15mins) , which can serve as a positive control for antibody performance.

What is the recommended protocol for using EIF4EBP1 (Ab-64) Antibody in immunohistochemistry?

For immunohistochemistry applications:

• Prepare paraffin-embedded tissue sections (4-6 μm thickness).

• Deparaffinize and rehydrate sections.

• Perform antigen retrieval (citrate buffer pH 6.0 is typically effective).

• Block endogenous peroxidase activity with hydrogen peroxide solution.

• Block non-specific binding with normal serum or protein blocking reagent.

• Incubate with EIF4EBP1 (Ab-64) Antibody at a dilution of 1:50 to 1:100 overnight at 4°C.

• Wash with PBS or TBS.

• Apply appropriate secondary antibody system.

• Develop with DAB or other chromogen.

• Counterstain, dehydrate, and mount .

The antibody has been validated for IHC in human colon carcinoma tissue, which could serve as a positive control sample .

What controls should be included when working with EIF4EBP1 (Ab-64) Antibody?

For rigorous experimental design, include:

Positive Controls:

• Jurkat cells treated with insulin (0.01U/ml, 15mins) for Western blot applications

• Human colon carcinoma tissue for IHC applications

• For cell-based ELISA: HeLa cells (typically express detectable levels of EIF4EBP1)

Negative Controls:

• Primary antibody omission control

• Isotype control (rabbit IgG at matching concentration)

• Non-expressing or knockdown cell lines

Normalization Controls:

• For Western blots: housekeeping proteins like GAPDH, β-actin, or tubulin

• For cell-based ELISA: GAPDH antibody is recommended for normalizing target absorbance values

• For phosphorylated EIF4EBP1 studies: include antibodies against total EIF4EBP1 for normalization

How can I distinguish between phosphorylated and non-phosphorylated forms of EIF4EBP1?

While the EIF4EBP1 (Ab-64) Antibody detects total EIF4EBP1 regardless of phosphorylation status, researchers often need to distinguish phosphorylation states:

• Western Blot Mobility Shift Analysis: Phosphorylated EIF4EBP1 migrates more slowly in SDS-PAGE than non-phosphorylated forms, appearing as multiple bands (α, β, γ bands) representing different phosphorylation states .

• Phospho-specific Antibodies: Use antibodies specific to particular phosphorylation sites:

  • Phospho-Thr45 antibodies

  • Phospho-Thr69 antibodies

  • Phospho-Ser111 antibodies

• Phosphatase Treatment: Treating samples with lambda phosphatase before Western blotting will collapse multiple bands into a single lower molecular weight band if differences are due to phosphorylation.

• Multiplexed Detection: Simultaneous use of phospho-specific and total EIF4EBP1 antibodies with different fluorescent tags can provide a phosphorylation ratio directly.

What transcription factors regulate EIF4EBP1 expression and how might this impact experimental design?

EIF4EBP1 transcription is regulated by multiple factors that should be considered in experimental design:

• MYC Oncoprotein: Directly stimulates EIF4EBP1 transcription and is often dysregulated in cancer models .

• Androgen Receptor: Regulates EIF4EBP1 expression in hormone-responsive tissues .

• Stress Response Regulators: ATF4 and ATF5 upregulate EIF4EBP1 in response to cellular stress .

• Hypoxia-Inducible Factor (HIF-1A): Activates EIF4EBP1 transcription under hypoxic conditions .

• E2F6: Acts as a transcriptional repressor, but paradoxically induces EIF4EBP1 promoter activity even at low expression levels .

• ETS1 and MYBL2: Both increase EIF4EBP1 promoter activity in a dose-dependent manner .

When designing experiments involving EIF4EBP1, consider:

• The cellular context and activation status of these transcription factors

• Whether treatments or disease states alter the expression of these regulators

• The potential for feedback loops, as EIF4EBP1 itself influences translation of these factors

How is EIF4EBP1 expression altered in cancer tissues, and what are the implications for using this antibody in cancer research?

EIF4EBP1 expression shows significant alterations in cancer with important implications for research:

What are the technical challenges when using EIF4EBP1 (Ab-64) Antibody in cell-based ELISA systems?

When using EIF4EBP1 (Ab-64) Antibody in cell-based ELISA systems, researchers should be aware of several technical considerations:

• Cell Type Selection:

  • Cell lines must express detectable levels of EIF4EBP1

  • For adherent cells, the protocol can be used directly

  • For suspension cells and loosely attached cells, additional steps are required:

    • Coat plates with 100 μL of 10 μg/mL Poly-L-Lysine for 30 minutes at 37°C before seeding cells

    • Use 8% formaldehyde (instead of 4%) for cell fixation

• Cell Number Optimization:

  • The optimal number of cells depends on EIF4EBP1 expression level, cell size, and treatment conditions

  • Cells should be 75-90% confluent at time of analysis

  • For HeLa cells, 30,000 cells per well is recommended

  • The assay can detect phospho-EIF4EBP1 in as few as 5,000 HeLa cells

• Normalization Methods:

  • GAPDH antibody is recommended as an internal positive control

  • Crystal Violet whole-cell staining can determine cell density to normalize for plating differences

  • For phosphorylated EIF4EBP1 studies, antibodies against total EIF4EBP1 should be used for normalization

• Technical Validation:

  • Include both positive and negative controls in the same plate

  • Perform each condition in duplicate or triplicate for statistical validity

How can phosphorylation status of EIF4EBP1 inform research on mTOR pathway activity in disease models?

EIF4EBP1 phosphorylation status serves as a key readout of mTOR pathway activity with significant implications for disease research:

• mTOR Signaling Indicator:

  • Hyperphosphorylated EIF4EBP1 indicates active mTOR signaling

  • EIF4EBP1 is directly phosphorylated by mTORC1 at multiple sites including Thr37/46, Ser65, and Thr70

  • Measuring EIF4EBP1 phosphorylation provides functional evidence of mTOR pathway activation

• Cancer Research Applications:

  • High EIF4EBP1 expression significantly correlates with enriched mTOR and cell proliferation-related gene sets in cancer

  • Gene set enrichment analysis (GSEA) of high EIF4EBP1-expressing tumors shows enrichment of MYC, G2M checkpoint, and E2F target genes

  • In breast cancer, EIF4EBP1 expression correlates with increased Ki67 expression and signaling via pharmacologically-activated mTOR gene sets

• Methodological Approach:

  • Use phospho-specific antibodies targeting key sites (Thr45, Thr69, Ser111) alongside total EIF4EBP1 antibodies

  • Calculate phosphorylation ratio (phospho/total) to normalize for expression level differences

  • Consider treatment with mTOR inhibitors (rapamycin or analogs) as negative controls

  • Insulin treatment (0.01U/ml, 15mins) serves as a positive control for mTOR activation

• Interpretation Framework:

  • Assess multiple phosphorylation sites, as the pattern of phosphorylation may be context-dependent

  • Consider feedback mechanisms that might affect results in different experimental conditions

  • In cancer studies, correlate phosphorylation status with patient outcomes and treatment responses

  • Remember that increased total EIF4EBP1 expression itself may reflect mTOR pathway deregulation

What are common issues when using EIF4EBP1 (Ab-64) Antibody and how can they be addressed?

How can I validate the specificity of EIF4EBP1 (Ab-64) Antibody in my experimental system?

Rigorous validation ensures reliable results and includes:

• Positive and Negative Controls:

  • Positive tissue/cell controls known to express EIF4EBP1 (Jurkat cells, human colon carcinoma)

  • Negative controls: primary antibody omission, isotype control

  • Stimulated vs. unstimulated samples (e.g., insulin treatment increases phosphorylation)

• Knockdown/Knockout Validation:

  • siRNA or shRNA against EIF4EBP1

  • CRISPR/Cas9-mediated knockout

  • Signal should be absent or significantly reduced in these systems

• Peptide Competition Assay:

  • Pre-incubate antibody with immunizing peptide

  • This should abolish or significantly reduce specific signal

• Orthogonal Detection Methods:

  • Verify with alternative antibodies targeting different epitopes

  • Correlate protein detection with mRNA expression data

  • Mass spectrometry validation of detected bands

• Phosphorylation-Specific Validation:

  • Treatment with phosphatase should eliminate phosphorylation-dependent signals

  • mTOR inhibitors should reduce phosphorylation at specific sites

  • Insulin or serum stimulation should increase phosphorylation

How can EIF4EBP1 (Ab-64) Antibody be used to study translational control in different disease models?

EIF4EBP1 (Ab-64) Antibody enables investigation of translational control mechanisms across various disease models:

• Cancer Research:

  • Monitor EIF4EBP1 expression and phosphorylation as a biomarker for cancer prognosis

  • Study treatment response to mTOR inhibitors by tracking EIF4EBP1 phosphorylation status

  • Investigate resistance mechanisms to targeted therapies by analyzing EIF4EBP1-regulated translation

• Neurological Disorders:

  • Study EIF4EBP1's role in medulloblastoma progression and relapse

  • Investigate translational control in models of neurodegeneration, where protein synthesis dysregulation is implicated

  • Analyze EIF4EBP1 status in relation to IDH mutation status in gliomas

• Metabolic Diseases:

  • Track EIF4EBP1 phosphorylation in response to insulin signaling alterations in diabetes models

  • Investigate nutrient-sensing pathways through EIF4EBP1 status

  • Explore connections between metabolic stress and translational control

• Experimental Approaches:

  • Combine EIF4EBP1 (Ab-64) Antibody with polysome profiling to correlate EIF4EBP1 status with translational efficiency

  • Use phospho-specific antibodies alongside total EIF4EBP1 detection to determine activation state

  • Perform immunoprecipitation to identify EIF4EBP1-interacting proteins in disease conditions

What analytical approaches are recommended for quantifying EIF4EBP1 expression in complex tissue samples?

For accurate quantification of EIF4EBP1 in complex tissues:

• Immunohistochemistry Quantification:

  • Use H-score system (0-300) calculated by multiplying staining intensity (0-3) by percentage of positive cells

  • Employ digital pathology software for unbiased quantification

  • Consider tissue microarrays for high-throughput analysis across multiple samples

• Cell-Type Specific Analysis:

  • Double immunofluorescence staining with cell-type markers

  • Single-cell RNA sequencing shows EIF4EBP1 is predominantly expressed in cancer epithelial cells, particularly in basal epithelial cell subclasses

  • Cell cybersorting via algorithms like xCell can help deconvolute expression patterns within the tumor microenvironment

• Expression Level Stratification:

  • For prognostic studies, consider quartile-based cutoffs for distinguishing high versus low expression

  • Median-based dichotomization has been used successfully in large cohort studies

  • Compare expression to matched normal tissues when available

• Correlation with Other Biomarkers:

  • Co-staining with Ki67 to assess proliferation correlation

  • Analyze in context of mTOR pathway activation markers

  • Consider tumor mutational burden, which has been shown to be highest in high EIF4EBP1-expression groups