EEF1A2 Antibody

What is EEF1A2 and why are antibodies against it important for research?

EEF1A2 is a protein that promotes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis. In humans, the canonical protein has 463 amino acids with a molecular weight of 50.5 kDa and is primarily localized in the nucleus . Unlike its ubiquitously expressed isoform EEF1A1, EEF1A2 shows tissue-specific expression patterns, notably in neural tissues like the caudate and cerebellum, as well as in skeletal muscle and heart .

EEF1A2 antibodies are critical research tools that enable detection, quantification, and characterization of this protein in various experimental contexts. They are particularly valuable for studying:

• Tissue-specific expression patterns

• Alterations in disease states (EEF1A2 is associated with developmental and epileptic encephalopathy )

• Post-translational modifications such as methylation

• Potential oncogenic roles (EEF1A2 is elevated in approximately 30% of tumor tissues )

What applications are EEF1A2 antibodies validated for?

When selecting an application, consider that antibody performance can vary significantly between applications, even with the same antibody. Always validate for your specific experimental conditions .

How do I select the appropriate EEF1A2 antibody for my experiments?

Selection criteria should include:

• Species reactivity: Confirm the antibody recognizes EEF1A2 in your species of interest. Commercial antibodies commonly react with human, mouse, and rat EEF1A2, with some also recognizing zebrafish, bovine, and other species .

• Application compatibility: Verify the antibody is validated for your intended application. Not all antibodies perform equally across different techniques .

• Epitope location: Consider where the antibody binds. For example, ab212172 recognizes an epitope within amino acids 200-300 , while others target different regions.

• Validation data: Review experimental validation, ideally including knockout/knockdown controls, which demonstrate specificity .

• Antibody format: Choose between polyclonal (broader epitope recognition) and monoclonal (higher specificity but potentially limited epitope access) .

• Post-translational modification detection: If studying modified forms of EEF1A2, select antibodies that specifically recognize these modifications .

How can I validate the specificity of EEF1A2 antibodies?

Comprehensive validation should include:

• Genetic validation: CRISPR/Cas9 knockout or RNAi knockdown of EEF1A2 should eliminate or significantly reduce signal. This provides the strongest evidence for specificity .

• Multiple antibody concordance: Results should be reproducible with antibodies targeting different epitopes of EEF1A2 .

• Tissue/cell type validation: Test the antibody in tissues with known EEF1A2 expression patterns (high in brain, skeletal muscle, heart; low in other tissues) .

• EEF1A1 cross-reactivity testing: Due to high sequence homology between these isoforms, confirm your antibody doesn't cross-react with EEF1A1 .

• Peptide competition: Pre-incubation with the immunizing peptide should abolish specific binding .

Example validation data from researchers demonstrated that bands recognized by EEF1A2 methyl-specific antibodies were depleted upon knockdown of their cognate lysine methyltransferases, confirming both enzyme-substrate relationships and antibody specificity .

What protocols yield optimal results for EEF1A2 detection in Western blots?

Positive control samples should include HEK-293 cells, MCF-7 cells, mouse brain tissue, or skeletal muscle tissue, all of which express detectable EEF1A2 levels .

How can I optimize immunohistochemistry protocols for EEF1A2 detection?

For successful IHC detection of EEF1A2:

• Fixation: Standard 10% neutral buffered formalin works well for most tissues .

• Antigen retrieval: Two effective methods have been reported:

  • TE buffer (pH 9.0) - primary recommendation

  • Citrate buffer (pH 6.0) - alternative method

• Antibody dilution: Start with 1:500-1:2000 and optimize based on signal-to-noise ratio .

• Detection systems: Both DAB (for brightfield) and fluorescence-based methods have been successfully used .

• Positive control tissues: Include human colon sections or neural tissues, which show reliable EEF1A2 expression .

IHC optimization should be performed systematically, changing one variable at a time while keeping others constant. Document all optimization steps thoroughly for reproducibility.

What are the methodological approaches for studying EEF1A2 methylation?

EEF1A2 undergoes significant post-translational methylation, which can be studied using:

• Methyl-specific antibodies: Researchers have developed antibodies that selectively recognize specific methylation states of EEF1A2, including:

  • K36me3

  • K55me2

  • K79me3

  • K165me2

  • K318me3

• Methyltransferase manipulation: CRISPR/Cas9-mediated knockdown of specific methyltransferases (eEF1AKMT4, METTL13, METTL21B, METTL10, N6AMT2) can reveal their roles in specific methylation events .

• Mass spectrometry validation: While more technically demanding, MS represents the gold standard for characterizing methylation events and should be used to validate antibody-based findings .

Research has revealed potential crosstalk between different methylation sites. For example, N6AMT2 depletion affected K36me3 levels, and METTL10 depletion impacted K79me3 levels, suggesting complex regulatory mechanisms .

How can I troubleshoot weak or non-specific signals when using EEF1A2 antibodies?

For IHC troubleshooting, additional considerations include:

• Optimize antigen retrieval by testing both TE buffer (pH 9.0) and citrate buffer (pH 6.0)

• Include positive control tissues in each experiment (human colon, brain tissue)

• Test a range of antibody dilutions, particularly if signal is weak or background is high

How should I interpret discordant results between different EEF1A2 antibodies?

When different antibodies targeting EEF1A2 yield conflicting results:

• Compare epitopes: Antibodies recognizing different epitopes may detect distinct conformations, isoforms, or post-translationally modified forms of EEF1A2 .

• Review validation data: Assess which antibody has more robust validation, particularly those validated with genetic approaches (knockout/knockdown) .

• Consider application differences: Some antibodies perform better in certain applications but poorly in others. For example, an antibody may work well for Western blot but not for IHC due to epitope accessibility .

• Evaluate fixation and preparation effects: Sample preparation can affect epitope accessibility. Different antibodies may require different antigen retrieval methods .

• Confirm with orthogonal methods: Use RNA-level analysis (RT-qPCR) or mass spectrometry to verify protein expression or modifications .

When publishing, report all antibodies used (including catalog numbers and dilutions) and acknowledge any discrepancies in the results obtained with different antibodies.

What controls are essential when using EEF1A2 antibodies in experimental studies?

For methylation studies, additional controls should include knockdown of specific methyltransferases to demonstrate the specificity of methyl-specific antibodies, as demonstrated in published research .

How can EEF1A2 antibodies be applied in cancer research?

EEF1A2 has been implicated in oncogenesis, with elevated expression in approximately 30% of tumor tissues and carcinoma cell lines . Research applications include:

• Expression profiling: EEF1A2 antibodies can be used to assess expression levels across tumor types and correlate with clinical parameters .

• Prognostic biomarker development: Evaluate the relationship between EEF1A2 expression and patient outcomes through IHC analysis of tissue microarrays.

• Mechanistic studies: Investigate how EEF1A2 contributes to oncogenic processes through:

  • Protein-protein interaction studies using co-immunoprecipitation

  • Post-translational modification analysis

  • Subcellular localization using immunofluorescence

• Therapeutic target validation: Assess the effects of EEF1A2 knockdown in conjunction with standard therapies or novel compounds.

When designing cancer studies, include appropriate normal tissue controls and consider the endogenous expression pattern of EEF1A2 to accurately interpret changes in cancer contexts.

What methodological approaches are recommended for studying EEF1A2 in neurodevelopmental disorders?

EEF1A2 has been associated with developmental and epileptic encephalopathy , making it relevant for neurodevelopmental research:

• Expression analysis in neural tissues: Use immunohistochemistry to map EEF1A2 expression during development and in disease states.

• Animal model validation: Confirm antibody cross-reactivity with model organisms (mouse, rat, zebrafish) to enable studies in genetic models.

• Functional studies in neurons: Combine EEF1A2 antibodies with functional assays to investigate:

  • Effects on protein synthesis in neurons

  • Interactions with neuronal proteins

  • Subcellular localization in different neuronal compartments

• Patient sample analysis: Compare EEF1A2 expression and post-translational modifications in patient-derived materials versus controls.

Researchers should prioritize antibodies with demonstrated specificity in neural tissues and consider the developmental timepoints relevant to the disorder being studied.

How can I quantitatively assess EEF1A2 methylation dynamics in aging or disease models?

Based on recent research showing changes in eEF1A methylation during aging , recommended approaches include:

• Longitudinal analysis: Use methyl-specific antibodies to track changes in EEF1A2 methylation across time points in:

  • Aging models

  • Disease progression models

  • Developmental time courses

• Multiplex immunodetection: Combine methylation-specific antibodies with markers of cellular processes to correlate methylation changes with functional outcomes.

• Methyltransferase activity assays: Complement antibody-based detection with assays measuring the activity of EEF1A2-specific methyltransferases.

• Quantitative image analysis: Apply digital pathology tools to quantify methylation-specific signals in tissue sections across experimental groups.

• Validation through mass spectrometry: Confirm antibody-based findings with MS-based quantification of methylation levels at specific residues.

Research has demonstrated that antibody-based detection can effectively reproduce data from more complex MS-based analyses, making antibodies valuable tools for monitoring these modifications .