EEF2KMT Antibody

What is EEF2KMT and what is its primary function in cells?

EEF2KMT (eukaryotic elongation factor 2 lysine methyltransferase) is a protein that catalyzes the trimethylation of eukaryotic elongation factor 2 (EEF2) specifically at lysine-525. This 36.9 kDa protein (330 amino acid residues in its canonical form) is localized in the cytoplasm and belongs to the EEF2KMT protein family. The enzyme is responsible for this critical post-translational modification that influences translational elongation during protein synthesis. Alternative splicing produces two different isoforms of this protein in humans. The protein is also known by several synonyms including family with sequence similarity 86 member A (FAM86A), putative protein N-methyltransferase FAM86A, and protein-lysine N-methyltransferase EEF2KMT .

What criteria should be used when selecting an EEF2KMT antibody for specific research applications?

When selecting an EEF2KMT antibody, researchers should consider:

• Application compatibility: Verify the antibody has been validated for your specific application (Western blot, ELISA, immunohistochemistry, etc.)

• Species reactivity: Ensure cross-reactivity with your experimental model organism

• Epitope location: Consider whether the antibody targets N-terminal, C-terminal, or internal regions

• Antibody format: Determine whether unconjugated or conjugated (biotin, HRP, FITC) antibody is appropriate

• Validation documentation: Request detailed validation data showing specificity for EEF2KMT

• Clone type: Consider polyclonal vs. monoclonal based on your specific research needs

The current market offers at least 40 EEF2KMT antibodies from 13 suppliers with varying specifications suitable for different experimental designs. Many antibodies target the C-terminal region, which may be beneficial for detecting full-length protein .

What validation methods should be implemented to confirm EEF2KMT antibody specificity?

For thorough validation, researchers should combine multiple approaches to establish specificity. Include positive controls (tissues/cells known to express EEF2KMT) and negative controls (tissues/cells with minimal expression). Knockdown studies using siRNA or CRISPR-Cas9 provide the most stringent validation by confirming signal reduction proportional to protein depletion .

What are the optimal protocols for using EEF2KMT antibodies in Western blotting applications?

For optimal Western blot detection of EEF2KMT:

• Sample preparation:

  • Extract proteins using RIPA or NP-40 lysis buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if investigating potential phosphorylation interactions

  • Quantify protein concentration using Bradford or BCA assay

• Gel electrophoresis:

  • Load 20-40 μg of total protein per lane

  • Use 10-12% polyacrylamide gels for optimal resolution of the 36.9 kDa protein

• Transfer and blocking:

  • Transfer to PVDF membrane (recommended over nitrocellulose for methylated proteins)

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

• Antibody incubation:

  • Dilute primary antibody 1:1000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Wash 3-5 times with TBST

  • Incubate with appropriate secondary antibody (typically 1:5000) for 1 hour

• Detection:

  • Develop using enhanced chemiluminescence

  • Expected band size: 36.9 kDa (canonical isoform)

  • Verify specificity with positive and negative controls

How can researchers effectively use EEF2KMT antibodies for immunohistochemistry and immunofluorescence?

For optimal immunohistochemistry/immunofluorescence using EEF2KMT antibodies:

• Tissue preparation:

  • Fix tissues in 4% paraformaldehyde

  • For FFPE sections, perform antigen retrieval using citrate buffer (pH 6.0) at 95-100°C for 15-20 minutes

  • For frozen sections, fix briefly in cold acetone

• Blocking and permeabilization:

  • Block endogenous peroxidase activity (for IHC) with 0.3% H₂O₂

  • Permeabilize with 0.1% Triton X-100 if detecting intracellular proteins

  • Block non-specific binding with 5-10% normal serum from the species of secondary antibody

• Antibody incubation:

  • Dilute primary antibody 1:100 (typical starting dilution, optimize as needed)

  • Incubate overnight at 4°C in a humidified chamber

  • Wash thoroughly with PBS or TBS (3-5 times, 5 minutes each)

  • Apply appropriate secondary antibody and develop using standard protocols

• Controls and validation:

  • Include tissue sections known to express EEF2KMT as positive controls

  • Include isotype controls and secondary-only controls

  • Validate cytoplasmic staining pattern consistent with known localization

How can EEF2KMT antibodies be utilized to investigate changes in methylation status during aging and cellular stress?

Recent research suggests that methylation of elongation factors may decline in aged tissues, potentially affecting translation efficiency. To investigate EEF2KMT activity and methylation dynamics:

• Aging studies methodology:

  • Compare young vs. aged tissue samples using quantitative Western blotting with both total EEF2 and methylation-specific antibodies

  • Calculate the ratio of methylated to total EEF2 to determine methylation status

  • Use mass spectrometry to confirm changes in methylation at specific residues

• Stress response investigation:

  • Subject cells to different stressors (oxidative stress, nutrient deprivation, heat shock)

  • Measure changes in EEF2KMT expression and activity

  • Correlate with global translation rates using puromycin incorporation assays

• Tissue-specific analysis:

  • Examine different tissues to identify variation in EEF2KMT activity

  • Muscle tissue shows notable changes in methylation patterns with age

  • Correlate changes with tissue-specific phenotypes of aging

This approach can reveal whether EEF2KMT activity and subsequent EEF2 methylation serve as biomarkers of aging or stress response, potentially opening new avenues for interventions targeting translation efficiency in age-related conditions.

What is the relationship between EEF2KMT activity and other post-translational modifications of translation factors?

Emerging evidence suggests complex regulatory networks involving multiple post-translational modifications of translation factors:

• Cross-talk investigation protocols:

  • Perform immunoprecipitation of EEF2 followed by mass spectrometry to map all modifications

  • Use specific antibodies against phosphorylated, methylated, and acetylated forms

  • Analyze sequential modifications using time-course experiments with pulse-chase labeling

• Interplay between modifications:

  • Knockdown studies of individual methyltransferases reveal that loss of one methylation event can affect others

  • Western blot analysis following knockdown of EEF2KMT can reveal compensatory changes in other modifications

  • Immunoprecipitation studies can identify protein complexes containing multiple modification enzymes

• Functional consequences:

  • Polysome profiling after manipulation of EEF2KMT activity

  • Ribosome footprinting to analyze translation efficiency

  • Correlation between modification patterns and mRNA-specific translation rates

Understanding this interplay provides insight into the complex regulation of translation elongation and potential therapeutic targets for conditions with dysregulated protein synthesis.

What are common pitfalls when using EEF2KMT antibodies and how can they be overcome?

How should researchers interpret quantitative changes in EEF2KMT levels or activity?

When quantitatively analyzing EEF2KMT expression or activity:

• Normalization approaches:

  • For Western blots, normalize to housekeeping proteins (β-actin, GAPDH)

  • For activity assays, establish standard curves with recombinant protein

  • When comparing tissues, consider tissue-specific reference genes

• Statistical analysis:

  • Perform at least three biological replicates for robust statistical analysis

  • Use appropriate statistical tests based on data distribution

  • Report fold changes with standard error and significance levels

• Biological interpretation considerations:

  • Small changes (1.5-2 fold) may be biologically significant for enzymes

  • Consider changes in substrate (EEF2) levels when interpreting activity changes

  • Evaluate effects on downstream pathways (translation rates, protein synthesis)

  • Correlate expression with methylation status of target lysine residues

• Methodological validation:

  • Confirm antibody linearity across the concentration range measured

  • Validate results using alternative detection methods (qPCR for mRNA, mass spectrometry for protein/activity)

How do age-related changes in EEF2KMT activity impact cellular protein synthesis capacity?

Recent research indicates that methylation levels of translation elongation factors, including EEF2, decline in aged tissues. This finding suggests:

• Translation efficiency changes:

  • Decreased EEF2 methylation correlates with reduced translation elongation rates

  • Age-dependent decline in protein synthesis quality control

  • Potential accumulation of mistranslated proteins contributing to proteostasis disruption

• Tissue-specific effects:

  • Muscle tissue shows pronounced changes in methylation patterns

  • Correlation between reduced EEF2 methylation and sarcopenia

  • Nervous system translation affected by methylation changes potentially contributing to neurodegeneration

• Interventional approaches:

  • Maintaining EEF2KMT activity might preserve translation fidelity during aging

  • Dietary or pharmacological interventions targeting methylation pathways

  • Exercise-induced restoration of methylation patterns in muscle tissue

These findings suggest EEF2KMT activity could be a potential biomarker for aging and a therapeutic target for age-related decline in protein synthesis capacity.

What techniques can be used to assess the specificity of EEF2KMT for its target lysine residue?

To investigate EEF2KMT target specificity:

• In vitro methylation assays:

  • Express recombinant EEF2 with site-directed mutagenesis at Lys-525

  • Perform in vitro methylation with purified EEF2KMT and radiolabeled methyl donor

  • Quantify methylation using scintillation counting or autoradiography

  • Analyze methylated products by mass spectrometry to confirm site specificity

• Cellular mutation studies:

  • Generate cell lines expressing EEF2 with K525R mutation (prevents methylation)

  • Compare methylation status using antibodies specific for methylated Lys-525

  • Analyze functional consequences on translation elongation rates

• Structural and computational approaches:

  • Model EEF2KMT-EEF2 interaction using available crystal structures

  • Identify key residues in the enzyme-substrate interface

  • Perform molecular dynamics simulations to predict specificity determinants

• Cross-substrate testing:

  • Test EEF2KMT activity on related elongation factors

  • Identify minimal peptide sequences that serve as substrates

  • Develop consensus motifs for recognition

These approaches can elucidate the molecular basis for the remarkable specificity of EEF2KMT for the Lys-525 residue of EEF2, potentially informing the development of specific inhibitors or activators.