EEF1B2 Antibody

What is EEF1B2 and why is it important in research?

EEF1B2 (also known as eEF1β or EF-1-beta) is a subunit of the eukaryotic translation elongation factor complex eEF1H. It functions primarily to stimulate the exchange of GDP bound to EF-1-alpha to GTP, facilitating protein translation. Recent research has revealed that EEF1B2 plays critical roles beyond translation, including regulation of bone marrow-derived mesenchymal stem cell (BMSC) differentiation by modulating the Wnt/β-catenin signaling pathway . This makes EEF1B2 antibodies essential tools for investigating cellular processes involving protein synthesis, cell differentiation, and various disease mechanisms.

What types of EEF1B2 antibodies are available for research and how should I select the appropriate one?

Several types of EEF1B2 antibodies are available for research purposes:

Selection should be based on:

• Your experimental application (WB, IHC, IF, etc.)

• Target species (human, mouse, rat)

• Need for specificity vs. sensitivity

• Validation in your experimental system

For reproducibility in long-term studies, monoclonal antibodies offer greater consistency, while polyclonal antibodies may provide higher sensitivity for low-abundance targets .

What is the molecular weight of EEF1B2 and why are there differences between calculated and observed weights?

EEF1B2 has a calculated molecular weight of approximately 24-29 kDa, but is typically observed at 30-34 kDa in SDS-PAGE analysis . This discrepancy is due to post-translational modifications, particularly phosphorylation. Research has identified that EEF1B2 can be phosphorylated by cyclin-dependent kinase 1 during mitosis, which affects its molecular weight and function . When interpreting Western blot results, researchers should expect to see bands in the 30-34 kDa range rather than at the calculated weight. If multiple bands appear, phosphatase treatment can help determine whether the additional bands represent phosphorylated forms.

What are the optimal conditions for Western blot detection of EEF1B2?

Based on validated protocols from multiple sources, the following optimized Western blot conditions are recommended:

For phosphorylation studies, it's critical to include phosphatase inhibitors (10 mM NaF, 20 mM β-glycerolphosphate, 1 mM sodium orthovanadate, 0.1 μM microcystin) in your lysis buffer to preserve EEF1B2 phosphorylation status .

How can I verify the specificity of my EEF1B2 antibody in Western blot applications?

To ensure specificity of EEF1B2 antibody detection:

• Positive controls: Use validated cell lines known to express EEF1B2, such as PC-3, HEK-293, HeLa, Jurkat, or SKOV-3 cells .

• Knockdown/knockout validation: Compare EEF1B2 detection in control vs. siRNA-treated or CRISPR-Cas9 knockout samples. Multiple studies have validated antibodies using EEF1B2 knockdown models .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to your membrane. Specific binding should be blocked.

• Multiple antibody validation: Use two antibodies raised against different epitopes of EEF1B2 to confirm consistent detection patterns.

• Molecular weight verification: Confirm band appearance at the expected molecular weight (30-34 kDa), accounting for post-translational modifications.

Researchers should also be aware of potential cross-reactivity with EEF1B2 pseudogenes in certain contexts .

What are the recommended protocols for EEF1B2 detection in tissue samples using immunohistochemistry?

For optimal immunohistochemical detection of EEF1B2 in tissue samples:

When working with mouse tissues in osteogenic research, it's advisable to use decalcification procedures that preserve antigenicity. Validated EEF1B2 antibodies have successfully detected expression in bone tissues following EDTA-based decalcification protocols .

How should I optimize immunofluorescence protocols for subcellular localization studies of EEF1B2?

For high-resolution subcellular localization studies of EEF1B2:

• Fixation: 4% paraformaldehyde for 10-15 minutes at room temperature provides optimal structural preservation while maintaining antigenicity.

• Permeabilization: Use 0.5% Triton X-100 for 10 minutes for adequate access to cytoplasmic EEF1B2 .

• Blocking: 5% BSA or normal serum for 30-60 minutes to reduce background.

• Antibody dilution: Begin with 1:50-1:200 dilution for primary antibody incubation. MCF-7 and HeLa cells have been validated as positive controls .

• Counterstaining: Use DAPI for nuclear visualization, which aids in determining EEF1B2's primarily cytoplasmic localization.

• Imaging: Confocal microscopy is recommended for precise subcellular localization. Z-stack acquisition enables 3D reconstruction of EEF1B2 distribution.

• Colocalization studies: Pair EEF1B2 antibodies with markers for specific organelles (ER, Golgi, ribosomes) to determine precise localization during different cellular processes.

For studies examining EEF1B2's role in translation complexes, co-staining with other eEF1H components (eEF1A, eEF1D, eEF1G) can provide valuable functional insights .

How can EEF1B2 antibodies be utilized to study its role in stem cell differentiation?

Recent research has established EEF1B2 as a critical regulator of bone marrow-derived mesenchymal stem cell (BMSC) differentiation . To investigate this role:

• Lineage-specific differentiation assays: During osteogenic or adipogenic differentiation, monitor EEF1B2 expression using validated antibodies (e.g., 10483-1-AP) at multiple time points (days 0, 3, 7, 14).

• Co-immunoprecipitation studies: Use EEF1B2 antibodies for co-IP to identify interaction partners during differentiation. This approach has revealed interactions with Wnt/β-catenin pathway components.

• Chromatin immunoprecipitation: Determine if EEF1B2 associates with chromatin regulatory elements using ChIP assays with anti-EEF1B2 antibodies.

• Knockdown/overexpression validation: Compare differentiation markers (ALP, RUNX2, OCN for osteogenesis; FABP4, LPL for adipogenesis) between control and EEF1B2 knockdown/overexpression conditions using Western blot and immunofluorescence.

• Time-course analysis: Monitor EEF1B2 phosphorylation status during differentiation using phospho-specific antibodies to determine if post-translational modifications correlate with lineage commitment.

RNA-seq analysis of EEF1B2 knockdown BMSCs has revealed that it regulates Wnt/β-catenin signaling, which functions as a molecular switch determining whether BMSCs differentiate into osteoblasts or adipocytes .

What methodologies are recommended for studying EEF1B2 phosphorylation during the cell cycle?

To study cell cycle-dependent phosphorylation of EEF1B2:

Research has shown that cyclin-dependent kinase 1 phosphorylates EEF1B2 during mitosis, reducing its affinity for eEF1A and affecting translation elongation .

How can I investigate the potential roles of EEF1B2 in cancer using antibody-based techniques?

To explore EEF1B2's roles in cancer:

• Tissue microarray analysis: Perform IHC on cancer tissue microarrays using validated EEF1B2 antibodies to quantify expression across multiple cancer types and correlate with clinical parameters. Human colon cancer, breast cancer, and pancreatic cancer tissues have been validated for IHC detection .

• Proximity ligation assay (PLA): Identify protein-protein interactions involving EEF1B2 that may be altered in cancer cells.

• RNA-immunoprecipitation (RIP): Investigate whether EEF1B2 associates with specific mRNAs in cancer cells using RIP followed by sequencing.

• Xenograft models: Apply IHC to detect EEF1B2 in tumor xenografts, as validated in U87 xenograft models .

• Multiplexed immunofluorescence: Co-stain for EEF1B2 and cancer markers to examine expression patterns within the tumor microenvironment.

• Patient-derived samples: Compare EEF1B2 expression between matched normal and cancer tissues using Western blot and IHC.

• Circulating tumor cell analysis: Explore whether EEF1B2 can be detected in circulating tumor cells using immunofluorescence.

Recent research indicates potential roles for EEF1B2 and its pseudogenes in various cancers, including non-small cell lung cancer, hepatocellular carcinoma, and acute myeloid leukemia .

What are common issues with EEF1B2 antibody applications and how can they be resolved?

For reproducible Western blots, the recommended dilution range for EEF1B2 antibodies is 1:500-1:10,000, with optimal results often achieved at 1:2000 . When using EEF1B2 antibodies for immunoprecipitation, 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate is recommended for efficient pulldown .

How should I validate EEF1B2 antibodies for detecting pseudogenes or splice variants?

EEF1B2 has several pseudogenes (EEF1B2P1, EEF1B2P2, EEF1B2P3) that may complicate antibody-based detection . To validate antibody specificity for EEF1B2 versus its pseudogenes:

• Epitope mapping: Determine if the antibody epitope is conserved in pseudogenes by sequence alignment. Request detailed epitope information from manufacturers.

• siRNA validation: Design siRNAs specifically targeting EEF1B2 but not pseudogenes, and verify antibody signal reduction.

• Mass spectrometry validation: After immunoprecipitation with anti-EEF1B2 antibodies, perform mass spectrometry to verify if peptides unique to EEF1B2 (and not pseudogenes) are detected.

• Recombinant protein controls: Test antibody reactivity against recombinant EEF1B2 and pseudogene proteins.

• Tissue-specific expression: Compare antibody staining patterns with known tissue-specific expression profiles of EEF1B2 versus its pseudogenes. For example, EEF1B2P2 transcripts are found in brain and muscle tissues .

• qPCR correlation: Correlate protein detection with qPCR using primers specific to either EEF1B2 or its pseudogenes.

For splice variant detection, design experiments that can distinguish the molecular weights of different variants and correlate immunoblotting results with RT-PCR or RNA-seq data.

How should researchers interpret discrepancies in EEF1B2 detection across different experimental platforms?

When encountering discrepancies in EEF1B2 detection between different methods:

• Expression level threshold differences: Western blot, IHC, and IF have different detection sensitivities. For low-abundance EEF1B2 expression, Western blot may detect signal while IHC appears negative.

• Epitope accessibility: Different fixation methods can mask epitopes. If discrepancies arise between fresh-frozen and FFPE samples, try multiple antigen retrieval methods. TE buffer (pH 9.0) is recommended for optimal retrieval in FFPE samples .

• Post-translational modifications: Phosphorylation affects antibody binding. In cell cycle studies, synchronize cells to ensure comparable modification status .

• Splice variants: EEF1B2 has multiple splice variants. Verify which variant your antibody detects and design experiments accordingly.

• Subcellular localization: EEF1B2 may relocalize during cellular processes. Use subcellular fractionation to complement immunofluorescence findings.

• Cross-reactivity profiles: Different antibodies have varying cross-reactivity with other EEF1 family members or pseudogenes. Validate using knockout/knockdown controls.

• Quantification methods: When comparing across platforms, normalize to appropriate controls (β-actin for Western blot, GAPDH or β-actin for IHC/IF) .

To resolve discrepancies, employ multiple antibodies targeting different epitopes and correlate protein detection with mRNA expression data.

What are the best practices for quantifying EEF1B2 expression in comparative studies?

For rigorous quantification of EEF1B2 expression:

• Western blot densitometry:

  • Use linear range of detection (avoid saturated signals)

  • Normalize to multiple loading controls (β-actin, GAPDH)

  • Include biological replicates (minimum n=3)

  • Present data as fold-change relative to control condition

  • Perform statistical analysis (t-test or ANOVA as appropriate)

• IHC quantification:

  • Use digital image analysis software for objective scoring

  • Establish clear positivity thresholds

  • Quantify both staining intensity and percentage of positive cells

  • Calculate H-scores or Allred scores for semi-quantitative analysis

  • Blind scorers to experimental conditions

• Immunofluorescence quantification:

  • Measure mean fluorescence intensity within defined regions

  • Perform background subtraction using negative control areas

  • Analyze multiple fields (>5) per condition

  • Use Z-stack projections for 3D samples

  • Consider subcellular distribution patterns

• Multiplexed analysis:

  • When comparing EEF1B2 with other markers, use multispectral imaging

  • Calculate colocalization coefficients (Pearson's or Mander's)

  • Normalize expression to cell number using nuclear counterstain

• Controls for quantification:

  • Include technical replicates to assess method variability

  • Use standard curves with recombinant protein when possible

  • Include positive control samples with known expression levels

  • For phosphorylation studies, include phosphatase-treated controls