IGF2BP2 Antibody

What is IGF2BP2 and why is it important in research?

IGF2BP2 is an RNA-binding protein that recruits target transcripts to cytoplasmic protein-RNA complexes (mRNPs), allowing for mRNA transport and transient storage. This "caging" mechanism modulates the rate and location at which target transcripts encounter the translational apparatus and protects them from degradation . IGF2BP2 preferentially binds to N6-methyladenosine (m6A)-containing mRNAs and increases their stability . Unlike IGF2BP1 and IGF2BP3, which are mainly oncofetal proteins, IGF2BP2 expression is maintained in many adult tissues, making it particularly relevant for studying post-transcriptional regulation in both normal and pathological states .

What are the common nomenclature variations for IGF2BP2?

Researchers should be aware of multiple nomenclature variations when searching literature:

• IGF2BP2 (official gene symbol)

• IMP-2 or IMP2

• VICKZ2

• IGF-II mRNA-binding protein 2

• Hepatocellular carcinoma autoantigen p62

Understanding these alternative names is crucial when designing literature searches or when interpreting antibody specificity information.

What is the molecular weight of IGF2BP2 and how does this inform antibody validation?

IGF2BP2 has a molecular weight of approximately 66.1 kilodaltons . This information is essential for Western blot validation, where researchers should confirm that their antibody detects a protein of the expected size. When validating a new IGF2BP2 antibody, always run positive controls (tissues/cells known to express IGF2BP2) alongside experimental samples and verify that the detected band matches this molecular weight. Variations from the expected molecular weight may indicate post-translational modifications, splice variants, or potential cross-reactivity with other proteins.

Which applications are IGF2BP2 antibodies typically validated for?

Based on commercial antibody specifications, IGF2BP2 antibodies are commonly validated for:

• Western Blot (WB)

• Immunohistochemistry on paraffin-embedded tissues (IHC-P) and frozen sections (IHC-fr)

• Immunocytochemistry (ICC)

• Immunofluorescence (IF)

• Immunoprecipitation (IP)

• ELISA

When selecting an antibody, researchers should ensure it has been validated for their specific application and target species, as reactivity can vary significantly between products.

What is the tissue expression pattern of IGF2BP2?

IGF2BP2 shows a complex tissue distribution pattern:

• Expressed in reproductive tissues: oocytes, granulosa cells, Leydig cells, spermatogonia, and semen

• Weakly expressed in heart, placenta, skeletal muscle, bone marrow, colon, kidney, salivary glands, testis, and pancreas

• Detected in fetal tissues: liver, ovary, gonocytes, and interstitial cells of the testis

• Present in certain cancer tissues, particularly in testicular cancer

This expression pattern should inform experimental design, particularly when selecting appropriate positive and negative control tissues.

How should researchers optimize antibody conditions for Western blot detection of IGF2BP2?

For optimal Western blot detection of IGF2BP2:

• Sample preparation:

  • Use RIPA or NP-40 buffer with protease inhibitors

  • Load 20-40 μg of total protein per lane

  • Denature samples at 95°C for 5 minutes in reducing conditions

• Antibody optimization:

  • Begin with manufacturer's recommended dilution (typically 1:500-1:2000)

  • Run a dilution series to determine optimal signal-to-noise ratio

  • Incubate primary antibody overnight at 4°C to improve sensitivity

  • Include detergents (0.05% Tween-20) in wash buffers to reduce background

• Detection considerations:

  • Use appropriate secondary antibody (typically anti-rabbit for polyclonal IGF2BP2 antibodies)

  • For low-expressing samples, consider more sensitive detection systems (enhanced chemiluminescence)

  • Expected band size is approximately 66 kDa

What critical controls should be included when using IGF2BP2 antibodies?

Rigorous experimental design requires multiple controls:

• Positive controls:

  • Cell lines or tissues with documented IGF2BP2 expression

  • Recombinant IGF2BP2 protein as a reference standard

• Negative controls:

  • Cell lines with IGF2BP2 knockdown/knockout

  • Secondary antibody-only controls to assess non-specific binding

  • Isotype controls to evaluate Fc-mediated interactions

• Specificity controls:

  • Peptide competition assays using the immunizing peptide

  • Comparative analysis with multiple antibodies targeting different epitopes

  • Controls for cross-reactivity with other IGF2BP family members (IGF2BP1, IGF2BP3)

These controls are essential for publication-quality research and should be documented in methods sections.

How can researchers use IGF2BP2 antibodies to study protein-RNA interactions?

To investigate IGF2BP2-RNA interactions:

• RNA Immunoprecipitation (RIP):

  • Crosslink protein-RNA complexes in live cells using formaldehyde or UV

  • Lyse cells in non-denaturing conditions to maintain complexes

  • Immunoprecipitate with anti-IGF2BP2 antibody

  • Extract and analyze bound RNAs by RT-PCR or sequencing

• Crosslinking Immunoprecipitation (CLIP):

  • Provides higher resolution mapping of binding sites

  • Use IGF2BP2 antibodies to pull down crosslinked protein-RNA complexes

  • This approach has revealed IGF2BP2 binding to transcripts like IGF2, MYC, and beta-actin

• Assessing m6A modification effects:

  • Combine with m6A-seq to correlate IGF2BP2 binding with methylation sites

  • Functional assays to assess how IGF2BP2 affects stability of m6A-modified targets

These approaches have revealed that IGF2BP2 binding is enhanced by m6A-modification, particularly in regions like the coding region instability determinant (CRD) of MYC mRNA .

What approaches can researchers use to study IGF2BP2 in cardiac disease models?

Based on recent findings about IGF2BP2's role in cardiac stress:

• Expression analysis in disease models:

  • Perform Western blot and IHC with IGF2BP2 antibodies on cardiac tissue samples during stress, remodeling, and recovery phases

  • Correlate with clinical parameters and outcome measures

• Transgenic model characterization:

  • Use IGF2BP2 antibodies to confirm expression levels in conditional, inducible mouse models

  • Track expression changes during disease progression and recovery

  • Correlate with functional cardiac parameters

• Subcellular localization studies:

  • Use immunofluorescence with IGF2BP2 antibodies to examine localization changes in stressed cardiomyocytes

  • Co-stain with markers for sarcomeres and mitochondria to assess relationship with these downregulated components

• Translational studies:

  • Apply similar approaches to human patient samples from DCM or myocardial infarction

  • Assess IGF2BP2 as a potential biomarker or therapeutic target

These approaches can help elucidate the mechanistic role of IGF2BP2 in cardiac pathology, where it has been shown to affect sarcomeric and mitochondrial proteins.

How can researchers use IGF2BP2 antibodies to explore its role in cancer progression?

For cancer research applications:

• Tissue microarray analysis:

  • Use validated IGF2BP2 antibodies for IHC on patient tissue arrays

  • Correlate expression with clinicopathological features and survival data

  • This approach has revealed that high IGF2BP2 expression predicts poor prognosis in HCC patients

• Mechanistic studies:

  • Perform co-immunoprecipitation to identify protein interaction partners in cancer cells

  • Use RIP-seq to identify cancer-specific RNA targets

  • Apply IGF2BP2 antibodies in functional studies following gene knockdown/overexpression

• Immune infiltration correlation:

  • Combine IGF2BP2 IHC with immune cell markers to assess relationship with tumor immune environment

  • This may inform combination therapy approaches with immune checkpoint inhibitors

• Therapeutic target validation:

  • Use antibodies to confirm target engagement in preclinical models testing IGF2BP2 inhibitors

  • Monitor expression changes in response to treatment strategies

What are common causes of false negative results with IGF2BP2 antibodies and how can they be addressed?

When facing detection failures:

• Sample preparation issues:

  • Protein degradation during extraction

  • Insufficient lysis or protein extraction

  • Solution: Use fresh protease inhibitors, optimize extraction protocols for specific tissue types

• Epitope accessibility problems:

  • Insufficient antigen retrieval in IHC/ICC

  • Solution: Test multiple antigen retrieval methods (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Extended retrieval times may be necessary for highly fixed tissues

• Sensitivity limitations:

  • Low endogenous expression levels

  • Solution: Use signal amplification methods like tyramide signal amplification

  • Consider more sensitive detection systems

  • Extend primary antibody incubation (overnight at 4°C)

• Antibody-specific issues:

  • Epitope masking due to protein-protein interactions

  • Post-translational modifications affecting antibody binding

  • Solution: Try alternative antibodies targeting different epitopes

How can researchers differentiate between IGF2BP family members with antibodies?

Distinguishing between IGF2BP1, IGF2BP2, and IGF2BP3 requires careful planning:

• Antibody selection:

  • Choose antibodies raised against unique regions with low sequence homology

  • Verify specificity against recombinant proteins of all three family members

  • Many commercial antibodies target the region between amino acids 500-C-terminus of IGF2BP2

• Validation approach:

  • Use siRNA knockdown of specific family members as controls

  • Compare expression patterns with known tissue distribution differences

  • In Western blots, carefully resolve proteins by molecular weight (though they are similar)

• Immunoprecipitation validation:

  • Perform IP followed by mass spectrometry to confirm identity

  • Use specific peptide competition assays

This differentiation is critical since the three IGF2BP proteins have overlapping but distinct functions and expression patterns.

What methodological considerations are important for immunohistochemical detection of IGF2BP2?

For optimal IHC results:

• Tissue preparation:

  • Proper fixation is critical (10% neutral buffered formalin for 24-48 hours)

  • Paraffin embedding should follow standard protocols

  • Use freshly cut sections (4-5 μm thick) for best results

• Antigen retrieval optimization:

  • Test both heat-induced epitope retrieval methods:

    • Citrate buffer (pH 6.0)

    • EDTA buffer (pH 9.0)

  • Optimize duration (typically 10-20 minutes)

• Blocking and antibody incubation:

  • Block endogenous peroxidase (3% H₂O₂) and non-specific binding (5% normal serum)

  • Optimize antibody concentration through titration

  • Most IGF2BP2 antibodies work best at 1:100-1:500 dilutions

  • Extend primary antibody incubation to overnight at 4°C for maximum sensitivity

• Signal detection considerations:

  • Use appropriate detection systems based on expected expression levels

  • For low expression, consider amplification systems (polymer-based or biotin-streptavidin)

  • Include proper controls on the same slide when possible

How can researchers investigate IGF2BP2's role in m6A RNA modification pathways?

To study IGF2BP2 as an m6A reader:

• Integrative experimental approach:

  • Combine m6A-seq to map modification sites

  • Perform IGF2BP2 RIP-seq or CLIP-seq to identify binding sites

  • Compare datasets to identify overlapping regions

• Functional validation:

  • Use reporter assays with wild-type vs. m6A-mutant binding sites

  • Assess RNA stability and translation efficiency

  • Manipulate m6A writers (METTL3/14) and assess effects on IGF2BP2 binding

• Structural studies:

  • Use purified proteins for in vitro binding assays

  • Compare binding kinetics between methylated and unmethylated RNA

These approaches have revealed that IGF2BP2 preferentially binds m6A-modified mRNAs, particularly affecting stability of targets like MYC .

What approaches can be used to confirm antibody specificity for IGF2BP2?

To ensure antibody specificity:

• Genetic approaches:

  • Test antibody in IGF2BP2 knockout/knockdown models

  • Compare signal in wild-type vs. modified samples

  • This provides the most definitive validation

• Biochemical validation:

  • Perform peptide competition assays with the immunizing peptide

  • Use multiple antibodies against different epitopes and compare results

  • Verify molecular weight matches the expected 66.1 kDa

• Bioinformatic assessment:

  • Analyze the antibody epitope for potential cross-reactivity

  • Check for sequence similarity with other proteins, particularly IGF2BP1 and IGF2BP3

• Expression pattern confirmation:

  • Verify that detected expression matches known tissue distribution

  • For example, confirm expression in reproductive tissues and weaker expression in heart, placenta, etc.

How can IGF2BP2 antibodies be used to investigate its role in hepatocellular carcinoma?

To investigate IGF2BP2 in HCC research:

• Expression analysis in patient cohorts:

  • Apply validated antibodies to tissue microarrays of HCC patients

  • Correlate expression with clinicopathological features

  • This approach has revealed that high IGF2BP2 expression predicts poor prognosis

• Autoantibody studies:

  • Use recombinant IGF2BP2 to detect autoantibodies in patient sera

  • Research has shown that 21% of HCC patients have autoantibodies against the p62 isoform of IGF2BP2

• Mechanistic investigations:

  • Use antibodies to monitor expression changes following experimental manipulation

  • Perform RIP-seq to identify cancer-specific RNA targets

  • Investigate relationship with key microRNA-regulated differentially expressed genes

• Therapeutic targeting:

  • Evaluate IGF2BP2 inhibition effects on cancer phenotypes

  • Assess combination with immune checkpoint inhibitors

  • Monitor tumor immune infiltration changes following IGF2BP2 manipulation

What experimental approaches can reveal IGF2BP2's role in cardiac pathophysiology?

Based on recent findings about IGF2BP2 in cardiac stress:

• Expression dynamics studies:

  • Use antibodies to track IGF2BP2 levels during:

    • Cardiac stress induction

    • Pathological remodeling phase

    • Recovery period

  • This approach has shown that IGF2BP2 is upregulated during stress and returns to normal during recovery

• Functional consequence analysis:

  • Correlate IGF2BP2 expression with:

    • Sarcomeric protein levels

    • Mitochondrial integrity

    • Cardiomyocyte structure

  • Studies have revealed that IGF2BP2 overexpression leads to downregulation of sarcomeric and mitochondrial proteins

• Translational investigations:

  • Apply similar approaches to human DCM or MI patient samples

  • Evaluate IGF2BP2 as a biomarker or therapeutic target

  • Research has confirmed IGF2BP2 upregulation in these patient populations

How can researchers investigate the relationship between IGF2BP2 and immune responses in disease?

To explore IGF2BP2's immunological connections:

• Correlation studies:

  • Use multiplex immunohistochemistry with IGF2BP2 and immune cell markers

  • Analyze spatial relationships between IGF2BP2-expressing cells and immune infiltrates

  • Research has shown that key genes regulated by IGF2BP2 are associated with immune cell infiltration in HCC

• Functional immunology:

  • Monitor immune responses following IGF2BP2 manipulation

  • Assess changes in cytokine production and immune signaling

  • Evaluate combination therapies targeting both IGF2BP2 and immune checkpoints

• Single-cell approaches:

  • Use single-cell RNA sequencing with antibody-based cell sorting

  • Identify cell populations with co-expression of IGF2BP2 and immune regulators

These approaches could support the hypothesis that IGF2BP2 inhibition may enhance immune responses to tumor cells .

What emerging applications for IGF2BP2 antibodies show promise in research?

Cutting-edge applications include:

• Spatial transcriptomics integration:

  • Combine IGF2BP2 immunofluorescence with in situ RNA detection

  • Map spatial relationships between IGF2BP2 protein and its target mRNAs

  • This can reveal tissue-specific post-transcriptional regulation patterns

• Mass cytometry (CyTOF) applications:

  • Develop metal-conjugated IGF2BP2 antibodies for high-dimensional analysis

  • Integrate with signaling and phenotypic markers

  • This approach can reveal single-cell heterogeneity in IGF2BP2 expression

• Proximity-based interaction studies:

  • Use IGF2BP2 antibodies in proximity ligation assays

  • Identify protein-protein interactions in situ

  • This can reveal context-specific interaction partners

• Therapeutic target validation:

  • Monitor IGF2BP2 expression changes during drug treatments

  • Assess efficacy of emerging IGF2BP2 inhibitors

  • These applications are particularly relevant for HCC and cardiomyopathies

How can researchers use IGF2BP2 antibodies to explore its role in RNA modification pathways?

To investigate IGF2BP2's role as an m6A reader:

• Integrative genomics approach:

  • Perform IGF2BP2 CLIP-seq using validated antibodies

  • Integrate with m6A-seq data

  • Identify overlapping modification and binding sites

  • Research has shown IGF2BP2 preferentially binds m6A-modified mRNAs

• Structure-function analysis:

  • Map the domains responsible for m6A recognition

  • Use antibodies against specific domains to disrupt binding

  • Assess functional consequences on target mRNA stability

• Target validation studies:

  • Focus on established targets like MYC where binding is enhanced by m6A-modification

  • Correlate IGF2BP2 binding with m6A levels and transcript stability

  • This approach has revealed IGF2BP2's role in stabilizing MYC mRNA through binding to its coding region instability determinant

• Pathway integration analysis:

  • Investigate relationships between IGF2BP2 and other m6A readers

  • Assess competition or cooperation between different readers

  • Examine how this affects downstream RNA fate decisions