CELF5 Antibody

What is CELF5 and why is it important in molecular biology research?

CELF5, also known as Bruno-like protein 5 or BRUNOL5, belongs to the CELF/BRUNOL protein family of RNA-binding proteins. It contains 2 adjacent N-terminal RNA recognition motif (RRM) domains and one C-terminal RRM domain, connected by an amino acid linker region of more than 160 amino acids . CELF5 is primarily expressed in all regions of fetal and adult brain with minimal expression elsewhere, making it particularly relevant for neurodevelopmental and neurological research . The protein plays a key role in regulating alternative splicing of pre-mRNA and may also be involved in mRNA editing and translation . Its study contributes to our understanding of post-transcriptional gene regulation mechanisms in the central nervous system.

How do I select the most appropriate CELF5 antibody for my research application?

Selecting the appropriate CELF5 antibody depends on your specific research application and target species. Consider these methodological factors:

For multi-species studies, verify cross-reactivity claims experimentally, as predictive reactivity (e.g., BLAST analysis) should be confirmed with empirical testing .

What is the difference between various epitope-targeted CELF5 antibodies?

Different epitope-targeted antibodies recognize distinct regions of the CELF5 protein, affecting specificity, sensitivity, and application suitability:

• Middle Region antibodies (e.g., ABIN2776548): Target central domains, offering broad reactivity across multiple species (human, mouse, rat, cow, dog, etc.) . The immunogen sequence "AFSGVQQYTAMYPTAAITPIAHSVPQPPPLLLQQQQREGPEGCNLFIYHLP" provides specific recognition .

• N-terminal targeted antibodies (e.g., A306998): Recognize amino acids 1-45, useful for identifying full-length protein but may miss truncated variants .

• Internal region antibodies (e.g., sc-138198): Target conserved internal sequences, offering good cross-species reactivity and reliability in multiple applications .

• Specific amino acid region antibodies (AA 323-372, AA 359-408): Provide precise epitope targeting, particularly valuable for distinguishing closely related family members or isoforms .

For research requiring isoform discrimination, epitope location relative to alternative splicing sites is crucial for accurate detection .

What are the optimal protocols for using CELF5 antibodies in Western blot applications?

For optimal Western blot results with CELF5 antibodies, follow this research-validated protocol:

• Sample preparation: Prepare tissue lysates (preferably from brain or neural tissues where CELF5 is highly expressed) using complete lysis buffers containing protease inhibitors .

• Electrophoresis conditions: Use 10-12% SDS-PAGE gels for optimal separation around the 52 kDa region where CELF5 migrates .

• Transfer conditions: For CELF5 (52 kDa), semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour to PVDF membranes is recommended .

• Blocking conditions: Block with 5% non-fat milk or BSA in TBS-T for 1-2 hours at room temperature .

• Primary antibody incubation: Dilute CELF5 antibodies as follows:

  • For rabbit polyclonals: 1:500-1:2,000

  • For goat polyclonals (e.g., T-12 sc-138198): starting dilution 1:200, range 1:100-1:1000

• Secondary antibody selection: For optimal results with CELF5 antibodies, use:

  • Donkey anti-goat IgG-HRP (for goat primaries) at 1:2000-1:5000

  • Anti-rabbit IgG-HRP (for rabbit primaries) at similar dilutions

• Expected results: Look for a specific band at approximately 52 kDa, with potential variation based on post-translational modifications or isoforms .

How should I optimize immunohistochemistry protocols for CELF5 detection in tissue samples?

Optimizing immunohistochemistry for CELF5 requires attention to several critical parameters:

• Tissue preparation: Use formalin-fixed, paraffin-embedded (FFPE) tissue sections with proper antigen retrieval methods. CELF5 has been successfully detected in human tonsil and thyroid cancer tissues .

• Antigen retrieval: Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) is typically effective for CELF5 antibodies. Optimize time (10-20 minutes) based on your specific antibody .

• Antibody dilution: Start with recommended dilutions:

  • For rabbit polyclonal IHC-validated antibodies: 1:40-1:200

  • For HPA042012 (Prestige Antibodies): 1:50-1:200

• Detection system: For best signal-to-noise ratio, use sensitive detection systems like polymer-based HRP systems rather than ABC methods .

• Controls: Always include:

  • Positive control tissues (human tonsil works well for CELF5)

  • Negative controls (primary antibody omission)

  • Peptide competition controls (when available) to verify specificity

• Expected staining pattern: CELF5 shows both nuclear and cytoplasmic localization. Evaluate staining in context of known expression patterns in brain tissue .

What validation methods should I use to confirm CELF5 antibody specificity?

To rigorously validate CELF5 antibody specificity, implement multiple complementary approaches:

• Peptide competition assays: Pre-incubate antibody with immunizing peptide (if available, like sc-138198 P) to confirm signal disappearance in target applications .

• siRNA/shRNA knockdown: Utilize available CELF5 siRNA (sc-97170) or shRNA Plasmid (sc-97170-SH) to verify reduced signal corresponds with reduced protein expression .

• Orthogonal method validation: Compare protein detection across multiple methods (WB, IHC, IF) using the same antibody .

• Multi-antibody approach: Use antibodies targeting different epitopes of CELF5 to confirm consistent detection patterns .

• RNA-seq correlation: For enhanced validation, correlate protein expression with RNA-seq data for CELF5 across tissues, as implemented in the Human Protein Atlas validation approach .

• Cross-reactivity assessment: Test for potential cross-reactivity with other CELF family members, particularly CELF3 and CELF6, which should show negative results with CELF5-specific antibodies .

How should I interpret variable CELF5 expression patterns across different tissue samples?

Interpreting variable CELF5 expression requires careful consideration of biological and technical factors:

• Tissue-specific expression: CELF5 is predominantly expressed in brain tissue with minimal expression elsewhere . When analyzing expression data:

  • High expression in brain regions is expected and biologically relevant

  • Expression in non-neural tissues may indicate either specialized functions or potential technical artifacts

• Developmental considerations: Compare expression between fetal and adult tissues, as CELF5 is expressed in both fetal and adult brain .

• Subcellular localization analysis: CELF5 localizes to both nucleus and cytoplasm , so differential compartment distribution may reflect functional states rather than antibody inconsistency.

• Quantification approaches: For comparing expression across samples:

  • In IHC: Use H-score or Allred scoring methods accounting for both intensity and percentage of positive cells

  • In WB: Normalize CELF5 signal to appropriate housekeeping proteins (β-actin for cytoplasmic, HDAC1 for nuclear fractions)

• Technical variation assessment: Evaluate fixation times, processing protocols, and storage conditions as potential sources of variability in IHC/IF results.

How do I reconcile contradictory results obtained with different CELF5 antibodies?

When faced with contradictory results from different CELF5 antibodies, employ this systematic analytical approach:

• Epitope mapping analysis: Document the specific epitopes recognized by each antibody and evaluate whether they target:

  • Regions affected by alternative splicing (potentially yielding isoform-specific detection)

  • Domains subject to post-translational modifications

  • Regions that may be masked in protein-protein or protein-RNA interactions

• Antibody validation comparison: Assess the validation depth for each antibody:

  • Enhanced validation antibodies (with orthogonal RNA-seq validation ) may be more reliable

  • Antibodies validated across multiple applications demonstrate higher reliability

• Species-specific considerations: Verify epitope conservation across species if working with non-human models .

• Methodology-dependent outcomes: Some antibodies perform better in certain applications:

  • Native vs. denatured conditions (WB vs. IP)

  • Fixed vs. frozen samples (paraffin IHC vs. frozen sections)

• Isoform detection: Determine if contradictory results stem from differential isoform detection, as CELF5 has at least two reported isoforms .

What statistical approaches are recommended for quantifying CELF5 expression changes in experimental studies?

For rigorous quantification of CELF5 expression changes, implement these statistical methodologies:

• Western blot densitometry analysis:

  • Use linear range validation to ensure quantification within the dynamic range

  • Normalize to multiple housekeeping proteins (not just one)

  • Apply ratio-metric analysis using reference standards across blots

  • Minimum n=3 independent biological replicates required for statistical validity

• IHC quantification methods:

  • Implement digital pathology analysis using whole slide imaging when possible

  • Quantify both staining intensity and percentage of positive cells

  • For brain tissue, analyze specific regions separately rather than averaging

• Statistical analysis recommendations:

  • For normally distributed data: paired t-tests for before/after comparisons, ANOVA for multiple group comparisons

  • For non-normally distributed data: non-parametric alternatives (Mann-Whitney, Kruskal-Wallis)

  • Report effect sizes (Cohen's d) in addition to p-values

  • Consider hierarchical/nested models for analyses with multiple sections per sample

• Multiple comparison correction:

  • Apply Benjamini-Hochberg FDR for multiple comparisons rather than simple Bonferroni

  • Report both corrected and uncorrected p-values for transparency

What are common causes of non-specific binding with CELF5 antibodies and how can they be minimized?

Non-specific binding with CELF5 antibodies can be systematically addressed using these research-validated approaches:

• Common sources of non-specificity:

  • Cross-reactivity with other CELF family members (particularly CELF3 and CELF6)

  • Fc receptor binding in immune cells

  • Endogenous biotin in tissues interfering with detection systems

  • Insufficient blocking leading to hydrophobic interactions

• Optimization strategies:

  • For Western blot: Increase blocking time/concentration (5% BSA instead of milk for phospho-detection), optimize antibody dilution (start with 1:500-1:1000 for polyclonals) , increase wash stringency with higher salt TBS-T.

  • For IHC/IF: Use protein-free blockers in high-background tissues, implement avidin-biotin blocking for biotin-based detection, use Fab fragments instead of whole IgG antibodies.

  • For all applications: Pre-absorb antibodies with tissue powder from species of interest.

• Antibody selection strategies:

  • When possible, select antibodies specifically non-cross-reactive with CELF3 and CELF6

  • Affinity-purified antibodies generally show higher specificity than crude antisera

  • Consider using secondary antibodies pre-adsorbed against multiple species

• Validation controls:

  • Always include peptide competition controls when possible

  • Use CELF5 knockout/knockdown tissues or cells as negative controls

  • Include isotype controls at equivalent concentrations to rule out non-specific binding

How can I adapt CELF5 antibodies for co-immunoprecipitation and protein-RNA interaction studies?

For successful adaptation of CELF5 antibodies to co-immunoprecipitation (co-IP) and RNA-protein interaction studies:

• Co-IP optimization for CELF5:

  • Use mild lysis conditions (NP-40 or CHAPS-based buffers) to preserve protein-protein interactions

  • Pre-clear lysates thoroughly to reduce non-specific binding

  • Use antibodies targeting epitopes away from protein interaction domains of CELF5

  • Recommended antibodies: CELF5 polyclonal targeting internal region (sc-138198) or middle region (ABIN2776548)

  • Cross-link antibodies to beads (using BS3 or DMP) to prevent IgG contamination in eluates

  • Include RNase treatment controls to distinguish RNA-dependent interactions

• RNA-IP (RIP) protocol adaptations:

  • Modify standard protocols with gentler crosslinking (0.1% formaldehyde for 10 minutes)

  • Include RNase inhibitors throughout all purification steps

  • For RNA binding studies, avoid antibodies targeting RRM domains of CELF5

  • Verify RIP enrichment using known CELF5 RNA targets (e.g., TNNT2 pre-mRNA)

  • Include appropriate negative controls (isotype antibody, non-target RNA)

• Proximity ligation assay (PLA) for in situ interaction detection:

  • Combine CELF5 antibody with antibodies against suspected interacting partners

  • Use rabbit anti-CELF5 with mouse antibodies against interaction partners to enable species-specific secondary detection

  • Optimize fixation to preserve both protein and RNA integrity

  • Include single antibody controls to verify specificity of interaction signals

What are effective strategies for multiplexing CELF5 detection with other neural markers?

For effective multiplexing of CELF5 with other neural markers, implement these methodological strategies:

• Immunofluorescence multiplexing approaches:

  • Sequential detection protocol: For antibodies from the same species, use sequential tyramide signal amplification (TSA) with microwave treatment between rounds

  • Multi-species approach: Combine rabbit anti-CELF5 with mouse antibodies against neural markers

  • Direct conjugation strategy: Consider having CELF5 antibodies directly conjugated to fluorophores to eliminate secondary antibody cross-reactivity

• Antibody selection for multiplexing:

  • For CELF5: Rabbit polyclonal antibodies at 1:50-1:200 dilution work well for IF

  • Pair with mouse monoclonals against neural markers

  • Verify that signal intensity between targets is balanced (may require titration)

• Spectral considerations:

  • Utilize spectrally distinct fluorophores with minimal bleed-through

  • Consider emission fingerprinting on confocal systems for closely overlapping signals

  • For brightfield multiplexing, use Opal or similar multicolor IHC systems

• Controls for multiplex experiments:

  • Single-stained controls for each antibody

  • Fluorescence-minus-one (FMO) controls to assess bleed-through

  • Absorption controls to verify antibody combinations don't interfere with each other

• Analysis approaches:

  • Implement colocalization analysis (Pearson's or Mander's coefficients)

  • Consider 3D analysis rather than single plane for volumetric assessment of colocalization

  • Use machine learning-based segmentation for automated quantification of multiple markers

How does CELF5 expression correspond with neural development and pathological conditions?

CELF5 expression patterns show significant biological relevance across neural development and pathological states:

• Developmental expression profile:

  • CELF5 is expressed in all regions of both fetal and adult brain , suggesting important roles in both developmental and mature neural functions

  • Unlike some developmental splicing factors that show temporal regulation, CELF5 appears to maintain expression throughout development

  • Research suggests potential roles in regulating splicing transitions during neural maturation

• Brain-region specificity:

  • CELF5 shows "little expression elsewhere" beyond brain tissues , highlighting its neural-specific functions

  • Further research using region-specific analyses may reveal more subtle expression patterns across brain structures

• Pathological associations:

  • Immunohistochemistry validation has demonstrated CELF5 detection in thyroid cancer tissue , suggesting potential dysregulation in certain cancer types

  • The role of CELF5 in neurological disorders remains an active area of investigation

  • As an RNA-binding protein regulating alternative splicing, CELF5 may influence splicing patterns of neurologically relevant transcripts

• Research gaps and opportunities:

  • Limited studies exist on CELF5 expression in neurodevelopmental disorders

  • Potential roles in regulating splice variants of ion channels or neurotransmitter receptors

  • Comparative analysis with other CELF family members in neural tissues

What methodological advances might improve CELF5 detection in low-expressing tissues?

For enhanced detection of CELF5 in tissues with low expression levels, consider these advanced methodological approaches:

• Signal amplification technologies:

  • TSA (Tyramide Signal Amplification) can significantly increase sensitivity for IHC/IF applications

  • RNAscope combined with IHC for correlative RNA-protein detection

  • Proximity ligation assay (PLA) for in situ protein detection with single-molecule sensitivity

• Sample preparation optimization:

  • Phosphatase inhibitors to preserve potential phosphorylated forms of CELF5

  • Optimized fixation protocols (shorter formalin fixation times, PAXgene fixation)

  • Antigen retrieval optimization specific to CELF5 epitopes

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM, PALM) for detection of low-abundance proteins

  • CODEX or other iterative imaging approaches for multiplexed detection

  • Digital pathology with computational enhancement

• Molecular engineering strategies:

  • Development of recombinant antibody fragments with enhanced tissue penetration

  • Nanobody-based detection systems specific for CELF5 epitopes

  • CRISPR-based endogenous tagging for live-cell imaging of CELF5

How can researchers effectively distinguish between CELF family members in experimental systems?

Distinguishing between CELF family members requires careful experimental design and specialized techniques:

• Antibody selection strategies:

  • Select antibodies targeting non-conserved regions between CELF family members

  • CELF5 (T-12) antibody is specifically documented as "non cross-reactive with CELF3 or CELF6"

  • Target unique domains outside the highly conserved RRM regions

  • When possible, validate with knockout/knockdown controls for each family member

• Expression analysis approaches:

  • Complement protein detection with transcript-specific analysis (RT-qPCR, RNA-seq)

  • Utilize isoform-specific primers to distinguish splice variants

  • Consider single-cell approaches to resolve mixed populations

• Functional discrimination methods:

  • RNA immunoprecipitation followed by sequencing (RIP-seq) to identify target RNA specificity

  • CLIP-seq approaches to map binding sites with nucleotide resolution

  • Splicing reporter assays to determine functional impact on alternative splicing

• Structural considerations:

  • Target epitopes in the divergent segment (160-230 aa) between RRM domains, which shows greater sequence variation between family members

  • Consider native conformation differences that may provide additional specificity

• Methodological controls:

  • Peptide competition with family-specific peptides

  • Parallel analysis of multiple family members

  • Correlation with known tissue-specific expression patterns (CELF5 being predominantly brain-specific)

What are the key performance differences between polyclonal and monoclonal CELF5 antibodies?

The choice between polyclonal and monoclonal CELF5 antibodies involves important performance tradeoffs:

For most research applications, rabbit polyclonal CELF5 antibodies provide the optimal balance of specificity and sensitivity, particularly for brain tissue analysis where CELF5 is predominantly expressed .

How should researchers evaluate conflicting manufacturer claims about CELF5 antibody performance?

When evaluating conflicting claims about CELF5 antibody performance, implement this critical assessment framework:

• Validation evidence assessment:

  • Examine the comprehensiveness of validation data provided:

    • Prestige Antibodies (powered by Atlas Antibodies) provide extensive IHC data across multiple tissues

    • Look for validation across multiple applications (WB, IHC, IF) rather than single-application testing

    • Assess whether orthogonal validation (RNA-seq correlation) is provided

  • Verify if knockout/knockdown controls were used in validation

  • Check for peptide competition controls demonstrating specificity

• Species reactivity verification:

  • Compare claimed reactivity with supporting evidence

  • Distinguish between predicted reactivity based on sequence homology vs. empirically tested reactivity

  • Consider testing in your specific model system regardless of claims

• Application-specific performance indicators:

  • Western blot: Look for clean bands at expected molecular weight (~52 kDa)

  • IHC: Evaluate staining pattern consistency with known localization (nuclear and cytoplasmic)

  • IF: Assess background levels and signal-to-noise ratio in validation images

• Independent validation resources:

  • Human Protein Atlas data for CELF5 antibody performance

  • Published literature using specific antibody clones

  • Consider benchmarking multiple antibodies in parallel in your experimental system

• Technical support responsiveness:

  • Manufacturers providing detailed technical protocols and troubleshooting

  • Availability of validation data beyond catalog information

  • Willingness to provide additional information about epitope specificity and validation methods