EFL1 Antibody

What is EFL1 and what biological functions does it serve?

EFL1 (Elongation factor-like GTPase 1) is a protein involved in the biogenesis of the 60S ribosomal subunit and translational activation of ribosomes. Together with SBDS, it triggers the GTP-dependent release of EIF6 from 60S pre-ribosomes in the cytoplasm, thereby activating ribosomes for translation by allowing 80S ribosome assembly and facilitating EIF6 recycling to the nucleus . EFL1 has low intrinsic GTPase activity that increases upon contact with 60S ribosome subunits . The protein is a member of the Classic translation factor GTPase protein family and has been associated with Shwachman-Diamond syndrome . The canonical protein has approximately 1120 amino acid residues with a molecular mass of 125.4 kDa, and up to 2 different isoforms have been reported .

What are common applications for EFL1 antibodies in research?

EFL1 antibodies are most frequently utilized in the following applications:

• Western Blot (WB): The most common application for detecting and quantifying EFL1 protein expression levels

• Immunohistochemistry (IHC): For visualizing EFL1 distribution in tissue sections, particularly in paraffin-embedded tissues

• ELISA: For quantitative detection of EFL1 in various sample types

• Immunocytochemistry and Immunofluorescence (ICC-IF): For examining subcellular localization
  Different antibodies may be optimized for specific applications, so researchers should select antibodies validated for their particular experimental needs.

What species reactivity is available for EFL1 antibodies?

Commercial EFL1 antibodies show reactivity with multiple species, reflecting the evolutionary conservation of this protein. Based on available products, reactivity has been confirmed or predicted for:

How do polyclonal and monoclonal EFL1 antibodies differ in research applications?

When selecting between polyclonal and monoclonal EFL1 antibodies, researchers should consider these key differences:
Polyclonal Antibodies:

• Recognize multiple epitopes on the EFL1 protein, potentially increasing detection sensitivity

• Offer more robust detection when protein conformation may be altered

• May show batch-to-batch variation that could affect reproducibility

• Examples include rabbit polyclonal antibodies targeting specific regions (N-terminal, C-terminal)
  Monoclonal Antibodies:

• Provide highly specific recognition of a single epitope

• Offer greater consistency between experiments and batches

• May be more sensitive to epitope masking or denaturation

• Generally produce lower background signal in applications like IHC and ICC
  For initial characterization or when protein expression is low, polyclonal antibodies may offer advantages in sensitivity. For quantitative or longitudinal studies requiring high reproducibility, monoclonal antibodies may be preferable .

What are critical factors for optimizing Western blot protocols with EFL1 antibodies?

Optimizing Western blot protocols for EFL1 detection requires attention to several key factors:
Sample Preparation:

• Complete cell lysis is essential as EFL1 associates with ribosomes

• Include protease inhibitors to prevent degradation of the 125.4 kDa protein

• Denature samples thoroughly at appropriate temperatures (typically 95-100°C for 5 minutes)
  Protocol Parameters:

• Gel selection: Use lower percentage gels (6-8%) for better resolution of the large EFL1 protein

• Transfer conditions: Extend transfer time for complete migration of higher molecular weight proteins

• Blocking: 5% BSA or non-fat dry milk in TBST is typically effective

• Primary antibody dilution: Start with manufacturer recommendations (often 1:1000) and optimize if needed

• Incubation: Overnight at 4°C often yields optimal results for specific detection
  Visualization:

• Enhanced chemiluminescence detection systems provide good sensitivity

• For the expected band size, look for ~125.4 kDa for the canonical protein, with possible additional bands representing isoforms

What is the recommended approach for immunohistochemical detection of EFL1?

For successful immunohistochemical detection of EFL1:
Tissue Preparation:

• Formalin-fixed paraffin-embedded (FFPE) tissues are commonly used

• Proper fixation time is critical to preserve epitopes while allowing antibody penetration
  Protocol Optimization:

• Antigen retrieval: Essential for FFPE samples, with heat-induced epitope retrieval in citrate buffer (pH 6.0) often effective

• Antibody dilution: Published protocols have utilized 1:20 dilution for some antibodies

• Incubation conditions: Extended primary antibody incubation (overnight at 4°C) may improve specific signal

• Detection system: HRP-conjugated secondary antibodies with DAB substrate provide good visualization
  Controls:

• Positive control: Human prostate tissue has been demonstrated to express detectable levels of EFL1

• Negative controls: Include sections with no primary antibody and isotype controls
  Researchers should be aware that optimization may be required for different tissue types and fixation methods .

How should EFL1 antibodies be stored and handled for maximum stability and performance?

Proper storage and handling of EFL1 antibodies is critical for maintaining their performance over time:
Storage Conditions:

• Short-term (up to 1 week): 2-8°C

• Long-term: -20°C in small aliquots to prevent freeze-thaw cycles

• Avoid repeated freeze-thaw cycles that can degrade antibody performance
  Buffer Composition:

• Many commercial preparations include stabilizers such as:

  • 0.09% (w/v) sodium azide as a preservative

  • 2% sucrose for cryoprotection
    Handling Precautions:

• Allow antibodies to equilibrate to room temperature before opening

• Centrifuge briefly before use to collect solution at the bottom of the vial

• Use sterile technique when handling to prevent contamination

• Return to appropriate storage conditions immediately after use
  Following these guidelines will help ensure consistent antibody performance across experiments .

How can researchers verify the specificity of EFL1 antibodies?

Verifying antibody specificity is crucial for experimental validity. For EFL1 antibodies, consider these validation approaches:
Experimental Validation Methods:

• Knockout/knockdown controls: Compare signal between wild-type samples and those with reduced/eliminated EFL1 expression

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

• Multiple antibody verification: Use antibodies targeting different EFL1 epitopes (N-terminal vs. C-terminal)

• Immunoprecipitation followed by mass spectrometry to confirm target identity
  Technical Controls:

• Isotype controls: Use the same concentration of non-specific antibodies of the same isotype

• Tissue controls: Include tissues known to express (positive) or lack (negative) EFL1

• Signal specificity: Verify expected molecular weight in Western blots (~125.4 kDa)
  Thorough validation ensures that observed signals genuinely represent EFL1 protein rather than non-specific binding or cross-reactivity.

What approaches are most effective for studying EFL1's interaction with SBDS and role in ribosome biogenesis?

To investigate EFL1's functional interactions:
Protein-Protein Interaction Studies:

• Co-immunoprecipitation using antibodies against EFL1 to pull down SBDS and other interacting partners

• Proximity ligation assays for in situ visualization of protein interactions

• FRET or BiFC approaches for studying interactions in living cells
  Functional Assays:

• GTPase activity assays to assess EFL1's enzymatic function and how it's modulated by SBDS

• Ribosome profiling to examine effects on translation

• Polysome analysis to evaluate 60S and 80S ribosome assembly
  Structural Approaches:

• Cryo-EM studies of EFL1 in complex with ribosomes and interaction partners

• Mutational analysis to identify critical domains for protein-protein interactions
  These methodologies can provide insights into the molecular mechanisms underlying EFL1's role in ribosome biogenesis and the pathogenesis of Shwachman-Diamond syndrome .

How should researchers design experiments to investigate EFL1 in the context of Shwachman-Diamond syndrome?

For disease-relevant studies:
Experimental Models:

• Patient-derived samples: Primary cells or tissues from affected individuals

• Cell line models: CRISPR/Cas9-engineered cell lines with disease-associated EFL1 mutations

• Animal models: Genetically modified mice with EFL1 mutations
  Analytical Approaches:

• Comparative expression analysis between patient and control samples

• Functional assays to assess ribosome biogenesis and protein synthesis rates

• Co-immunoprecipitation studies to examine altered protein interactions

• Rescue experiments: Introducing wild-type EFL1 into mutant cells to restore function
  Translational Relevance:

• Focus on tissue-specific effects, particularly in bone marrow and pancreas

• Correlate molecular findings with clinical phenotypes

• Consider therapeutic strategies targeting the EFL1 pathway
  Such comprehensive approaches can provide insights into disease mechanisms and potentially identify therapeutic targets .

How should researchers address weak or absent signal when using EFL1 antibodies?

When encountering weak or absent signals:
Sample Considerations:

• Verify EFL1 expression level in your sample (reported to be low in brain tissue)

• Ensure adequate protein loading (consider 2-3x standard amounts for low-abundance proteins)

• Check sample preparation method (complete lysis, denaturation, protein degradation)
  Protocol Modifications:

• Increase antibody concentration or incubation time

• Enhance antigen retrieval for IHC/ICC applications

• Use signal amplification methods (HRP polymers, TSA systems)

• Extend exposure time for Western blots
  Antibody and Reagent Quality:

• Use fresh antibody aliquots to avoid decreased activity from freeze-thaw cycles

• Verify antibody storage conditions and expiration dates

• Test a different lot or source of antibody

• Check the reactivity profile matches your experimental species
  Systematic troubleshooting can help determine whether the issue relates to technique, reagents, or biological factors .

What strategies can resolve non-specific binding issues with EFL1 antibodies?

To reduce background and non-specific binding:
Blocking Optimization:

• Test different blocking agents (BSA, normal serum, commercial blockers)

• Increase blocking time or concentration

• Use the blocking agent in antibody dilution buffers
  Washing Improvements:

• Increase number and duration of wash steps

• Use more stringent washing buffers (higher salt concentration or detergent)

• Ensure thorough washing between each step
  Antibody Considerations:

• Titrate primary and secondary antibodies to optimal concentrations

• Pre-absorb antibodies with non-specific proteins

• Consider using more specific monoclonal antibodies if polyclonals show high background

• Use blocking peptides to confirm specificity of observed signals
  Detection System:

• Reduce substrate incubation time

• Use detection systems with lower background characteristics

• Consider fluorescent-based detection for better signal-to-noise ratios
  These adjustments can significantly improve signal specificity and experimental clarity .

How should researchers interpret changes in EFL1 expression across different experimental conditions?

For proper interpretation of EFL1 expression changes:
Quantification Approach:

What controls are essential when investigating post-translational modifications of EFL1?

For studying EFL1 post-translational modifications (PTMs):
Essential Controls:

• Treatment controls: Samples with modification-inducing or inhibiting treatments

• Specificity controls: Antibodies specifically recognizing modified forms

• Enzymatic verification: Treatment with phosphatases, deglycosylases, etc., to confirm modification type

• Positive controls: Samples known to contain the modification of interest
  Technical Approaches:

• Immunoprecipitation followed by PTM-specific detection

• Mass spectrometry for comprehensive PTM mapping

• 2D gel electrophoresis to separate modified forms

• Site-directed mutagenesis to confirm modification sites
  Analytical Considerations:

• Compare conditions that might alter modification status (cell cycle, stress, disease states)

• Investigate the impact of signaling pathway modulators

• Correlate modifications with functional outcomes
  These strategies help ensure that observed modifications are specific and biologically relevant .

How can researchers incorporate new fusion protein technologies with EFL1 antibodies?

The development of novel fusion protein technologies offers exciting possibilities for EFL1 research:
Innovative Applications:

• Antibody fusion proteins to enhance stability and detection sensitivity

• CRISPR-based tagging of endogenous EFL1 for live-cell imaging

• Nanobody-based detection systems for improved tissue penetration

• Proximity-dependent labeling (BioID, APEX) to identify novel EFL1 interaction partners
  The recent advances in engineering antibodies with fusion proteins have demonstrated increased stability during immunization processes, which could be applied to generate more effective EFL1 antibodies . These technologies offer promising approaches for enhancing both the generation and application of antibodies for studying challenging targets like EFL1.

What are future perspectives for multiplexing EFL1 detection with other ribosome biogenesis markers?

Multiplexed detection approaches offer comprehensive insights into ribosome biology:
Technical Approaches:

• Multiplex immunofluorescence with antibodies raised in different host species

• Sequential immunostaining protocols for co-detection of multiple targets

• Mass cytometry (CyTOF) for highly multiplexed protein detection

• Spatial transcriptomics combined with protein detection
  Experimental Considerations:

• Selection of compatible antibodies to avoid cross-reactivity

• Rigorous controls for each target in the multiplex panel

• Spectral unmixing for fluorophores with overlapping emission spectra

• Automated image analysis for quantitative assessment
  Research Applications:

• Simultaneous monitoring of multiple ribosome biogenesis factors

• Co-localization studies of EFL1 with interaction partners

• Correlation of EFL1 with disease markers in clinical samples

• System-level analysis of ribosome assembly dynamics
  These approaches can provide integrated views of ribosome biogenesis regulation in normal and pathological conditions, advancing our understanding of EFL1's biological roles .