IGF2BP1 Antibody, FITC conjugated

What is IGF2BP1 and what is its significance in cellular function?

IGF2BP1 (Insulin-like Growth Factor 2 mRNA Binding Protein 1) is a critical m6A (N6-methyladenosine) reader protein that recognizes m6A target transcripts and influences cancer development through post-transcriptional regulation. The protein consists of six canonical RNA-binding domains, including four KH (K homology) domains and two RRM (RNA recognition motifs) domains, with the KH domains playing a major role in facilitating RNA binding . IGF2BP1 has a calculated molecular weight of 63 kDa (577 amino acids) but is typically observed at 65-70 kDa in experimental conditions . The protein is involved in stabilizing various mRNA targets in an m6A-dependent manner, and its aberrant overexpression is associated with poor prognosis in several cancer types, particularly hepatocellular carcinoma, gallbladder cancer, and breast cancer .

What are the key specifications of FITC-conjugated IGF2BP1 antibody?

The FITC-conjugated IGF2BP1 antibody is designed for fluorescence-based detection applications with the following specifications:

The fluorescein isothiocyanate (FITC) conjugation facilitates direct visualization in fluorescence-based experiments without requiring secondary antibodies .

What are the optimal storage conditions for maintaining antibody activity?

To maintain the activity and integrity of the FITC-conjugated IGF2BP1 antibody, follow these storage protocols:

• Aliquot upon receiving to minimize freeze-thaw cycles

• Store at -20°C

• Protect from light exposure, as FITC is photosensitive

• Avoid repeated freeze/thaw cycles which can degrade both the antibody and the fluorophore

• For short-term storage, keep in buffer containing 0.01 M PBS, pH 7.4, with 0.03% Proclin-300 and 50% Glycerol

Improperly stored antibodies may exhibit reduced binding efficiency, increased background, or complete loss of signal in imaging applications. The antibody should remain stable for at least one year when stored properly at -20°C .

What are the recommended dilutions for different experimental applications?

The optimal working dilutions vary depending on the specific application:

It is crucial to titrate the antibody in each experimental system to obtain optimal results, as the required concentration may vary based on sample type, fixation method, and detection system .

How should I design and optimize immunofluorescence experiments using this antibody?

For optimal immunofluorescence experiments with FITC-conjugated IGF2BP1 antibody:

• Sample preparation: Fix cells using 4% paraformaldehyde for 15-20 minutes at room temperature. For tissue sections, an antigen retrieval step using TE buffer (pH 9.0) is recommended.

• Blocking: Use 5-10% normal serum (from the same species as the secondary antibody would be, if not using a directly conjugated antibody) with 0.1-0.3% Triton X-100 for 1 hour to reduce non-specific binding.

• Antibody dilution: Start with a 1:400 dilution for the FITC-conjugated IGF2BP1 antibody and optimize as needed. Incubate overnight at 4°C in a humidified chamber.

• Controls:

  • Negative control: Omit primary antibody

  • Isotype control: Use rabbit IgG-FITC at the same concentration

  • Positive control: Use A375 cells, which have been validated for this antibody

• Counterstaining: Use DAPI (1:1000) for nuclear staining, avoiding fluorophores that overlap with FITC's emission spectrum (515 nm).

• Mounting: Use an anti-fade mounting medium specifically designed for fluorescent samples to prevent photobleaching.

• Microscopy parameters: Use the 488 nm laser line for excitation and collect emission around 515 nm, adjusting exposure settings to prevent photobleaching while maintaining adequate signal intensity .

What positive controls and validated cell lines should I use for IGF2BP1 detection?

The following cell lines and tissues have been validated for IGF2BP1 detection:

Western Blot (WB) positive controls:

• HEK-293 cells

• Jurkat cells

• HepG2 cells

• HuH-7 cells

• A375 cells

• Mouse kidney tissue

• Rat kidney tissue

Immunoprecipitation (IP) positive controls:

• HEK-293 cells

Immunohistochemistry (IHC) positive controls:

• Human lung cancer tissue

• Human liver cancer tissue

Immunofluorescence (IF/ICC) positive controls:

• A375 cells

When establishing a new experimental system, using these validated samples as positive controls can help confirm proper antibody functionality before proceeding to experimental samples.

How does IGF2BP1 function in the tumor immune microenvironment (TIME)?

IGF2BP1 plays a significant role in regulating the tumor immune microenvironment through several mechanisms:

• Immune cell infiltration: IGF2BP1 knockdown induces cancer cell apoptosis, which subsequently activates immune cell infiltration, including CD4+ and CD8+ T cells, CD56+ NK cells, and F4/80+ macrophages in tumor tissues .

• PD-L1 regulation: IGF2BP1 influences the expression of PD-L1, an important immune checkpoint. Knockdown of IGF2BP1 significantly suppresses PD-L1 expression, potentially enabling better immune surveillance .

• Target mRNA stability: As an m6A reader, IGF2BP1 regulates the stability of target mRNAs like c-MYC, which are associated with cell apoptosis and immune response .

• Tumor progression: In hepatocellular carcinoma (HCC) models, IGF2BP1 dysfunction leads to antitumor immunity by recruiting tumor-infiltrating immune cells and blocking immunosuppressive factor expression .

These findings suggest that targeting IGF2BP1 may serve as a novel strategy for cancer immunotherapy by reshaping the tumor immune microenvironment. Using FITC-conjugated IGF2BP1 antibodies in multicolor flow cytometry or immunofluorescence microscopy can help researchers visualize and quantify these immune cell interactions in complex tissue environments.

What is the significance of IGF2BP1's allosteric regulation in cancer research?

The allosteric regulation of IGF2BP1 represents a significant advancement in cancer therapeutic approaches:

• Structural basis: IGF2BP1 contains KH domains that are crucial for RNA binding. The discovery that small molecules like cucurbitacin B (CuB) can bind to IGF2BP1 at a unique site (Cys253) in the KH1-2 domains reveals an allosteric regulatory mechanism .

• Conformational changes: CuB binding induces conformational changes in IGF2BP1's KH1-2 domains, as evidenced by decreased tryptophan fluorescence intensity and reduced protein helicity. These changes disrupt IGF2BP1's ability to recognize and bind m6A-modified mRNA targets .

• Functional consequences: This allosteric regulation blocks IGF2BP1 recognition of m6A mRNA targets such as c-MYC, leading to decreased target mRNA stability, increased cancer cell apoptosis, and enhanced immune response .

• Therapeutic potential: Targeting IGF2BP1 allosterically offers a novel approach to cancer treatment by simultaneously inducing tumor cell apoptosis and recruiting immune cells to the tumor microenvironment, as well as blocking PD-L1 expression .

Using FITC-conjugated IGF2BP1 antibodies in combination with confocal microscopy and fluorescence resonance energy transfer (FRET) techniques could help visualize these conformational changes and protein-protein interactions in living cells, providing deeper insights into the mechanisms of allosteric regulation.

How can FITC-conjugated IGF2BP1 antibodies be used in RNA-protein interaction studies?

FITC-conjugated IGF2BP1 antibodies offer valuable tools for investigating RNA-protein interactions through several methodologies:

• RNA Immunoprecipitation (RIP): FITC-conjugated IGF2BP1 antibodies can be used in RIP assays to isolate and identify RNA targets bound to IGF2BP1 in vivo. After crosslinking RNA-protein complexes, the antibody can pull down IGF2BP1 along with its bound RNAs, which can then be analyzed by RT-qPCR or RNA sequencing .

• Fluorescence microscopy of RNA granules: IGF2BP1 is known to form RNA granules. Using FITC-conjugated antibodies in fixed or permeabilized cells allows visualization of these granules and their dynamics, especially when combined with RNA FISH (Fluorescence In Situ Hybridization) for specific target RNAs.

• Co-localization studies: The FITC conjugation enables direct visualization of IGF2BP1 co-localization with other proteins involved in m6A recognition or RNA metabolism, when combined with antibodies conjugated to spectrally distinct fluorophores.

• FRAP (Fluorescence Recovery After Photobleaching): By transfecting cells with IGF2BP1 fused to a red fluorescent protein and staining with FITC-conjugated anti-IGF2BP1 antibodies, researchers can perform dual-color FRAP experiments to study the dynamics of both newly synthesized and existing IGF2BP1 populations in RNA granules.

• Single-molecule tracking: Advanced microscopy techniques combined with FITC-conjugated antibodies can enable tracking of individual IGF2BP1 molecules and their interaction with RNA in live cells, providing insights into binding kinetics and spatial organization.

When designing these experiments, it's important to consider that the antibody's epitope (aa 440-534) is within the KH domains region, which may interfere with RNA binding in certain experimental contexts .

What are common issues encountered with FITC-conjugated antibodies and how can they be resolved?

When working with FITC-conjugated IGF2BP1 antibodies, researchers may encounter several common issues:

• Photobleaching: FITC is relatively prone to photobleaching.

  • Resolution: Use anti-fade mounting media, minimize exposure to light during preparation, reduce exposure time and laser intensity during imaging, and consider taking images of control samples last.

• Autofluorescence: Biological samples may exhibit background fluorescence in the FITC channel.

  • Resolution: Include proper negative controls, use longer fixation times to reduce autofluorescence, consider tissue autofluorescence quenching reagents, and adjust imaging settings to minimize background.

• pH sensitivity: FITC fluorescence is sensitive to pH changes.

  • Resolution: Maintain consistent pH (ideally 7.0-8.0) in all buffers and mounting media to ensure optimal fluorescence intensity.

• Signal variability: Inconsistent staining patterns between experiments.

  • Resolution: Standardize fixation protocols, antibody concentration, incubation time and temperature, and imaging parameters. When comparing experimental groups, process and image all samples in parallel.

• Cross-reactivity: Non-specific binding leading to false positive signals.

  • Resolution: Include proper blocking steps (5-10% normal serum), validate antibody specificity using IGF2BP1 knockout/knockdown samples, and compare staining patterns with literature reports.

• Low signal-to-noise ratio: Weak specific signal relative to background.

  • Resolution: Optimize antibody concentration, increase incubation time (overnight at 4°C), enhance antigen retrieval methods, and consider signal amplification techniques if necessary.

How can I verify the specificity of IGF2BP1 antibody detection in my experimental system?

To verify the specificity of the FITC-conjugated IGF2BP1 antibody in your experimental system:

• Knockout/knockdown validation: Use IGF2BP1 knockout or knockdown samples as negative controls. The 22 published KD/KO studies using this antibody provide a strong foundation for this approach .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (aa 440-534 of human IGF2BP1) before application to samples. This should abolish specific staining if the antibody is truly specific.

• Multiple antibody comparison: Compare staining patterns with different IGF2BP1 antibodies recognizing distinct epitopes to confirm consistent localization and expression patterns.

• Western blot correlation: Perform western blotting on the same samples used for immunofluorescence to confirm that protein levels correlate between techniques. The expected molecular weight should be 65-70 kDa .

• Cross-species validation: Test the antibody in samples from different species (human, mouse, rat) where IGF2BP1 is conserved to confirm expected reactivity patterns .

• RNA-dependency test: Since IGF2BP1 is an RNA-binding protein, treating samples with RNase before immunostaining can reveal whether the detected localization is RNA-dependent.

• Positive control validation: Always include known positive controls such as A375 cells, HEK-293 cells, or HepG2 cells, which have been validated for this antibody .

How should I interpret changes in IGF2BP1 localization in response to experimental treatments?

Interpreting changes in IGF2BP1 localization requires consideration of its biological functions and known response patterns:

• Nuclear-cytoplasmic shuttling: IGF2BP1 can shuttle between the nucleus and cytoplasm. An increase in nuclear localization might indicate active involvement in early mRNA processing, while cytoplasmic accumulation suggests roles in mRNA transport, localization, or translation.

• Granular structures: IGF2BP1 often appears in distinct cytoplasmic granules. Changes in granule size, number, or intensity may indicate altered RNA processing activities or stress responses.

• Co-localization patterns: Changes in co-localization with processing bodies (P-bodies), stress granules, or other RNA-binding proteins can indicate shifts in IGF2BP1's functional associations.

• Treatment-specific responses:

  • Cell stress conditions often lead to recruitment of IGF2BP1 to stress granules

  • Cell cycle changes may alter distribution patterns

  • Drug treatments targeting m6A pathways may disrupt IGF2BP1 binding to its RNA targets

• Cancer-related changes: In cancer contexts, altered IGF2BP1 localization may correlate with changes in immune cell infiltration or PD-L1 expression as observed in HCC models .

• Allosteric modulator effects: Treatments with compounds like cucurbitacin B may cause conformational changes in IGF2BP1 that affect its localization pattern by altering RNA-binding capabilities .

When analyzing such changes, quantitative approaches (measuring nuclear/cytoplasmic ratios, granule numbers, co-localization coefficients) provide more reliable data than qualitative observations alone. Time-course experiments can also reveal the dynamics of IGF2BP1 relocalization in response to treatments.

How is IGF2BP1 being investigated as a therapeutic target in cancer research?

IGF2BP1 is emerging as a promising therapeutic target in cancer research through several innovative approaches:

• Allosteric regulation: The discovery that small molecules like cucurbitacin B (CuB) can allosterically regulate IGF2BP1 by binding to Cys253 in its KH1-2 domains represents a novel therapeutic strategy. This approach disrupts IGF2BP1's ability to stabilize oncogenic mRNAs like c-MYC .

• Immune microenvironment modulation: IGF2BP1 inhibition induces cancer cell apoptosis, which subsequently activates immune cell infiltration (CD4+, CD8+ T cells, CD56+ NK cells, and F4/80+ macrophages) while decreasing PD-L1 expression. This dual effect makes it an attractive target for combination with immunotherapies .

• m6A pathway targeting: As a critical m6A reader, IGF2BP1 represents one node in the broader m6A epitranscriptomic network. Therapies targeting IGF2BP1 could disrupt cancer-promoting m6A-dependent mRNA stabilization .

• Cancer-specific overexpression: IGF2BP1's overexpression in multiple cancer types (particularly hepatocellular carcinoma, gallbladder cancer, and breast cancer) with limited expression in normal adult tissues makes it an attractive target with potentially reduced side effects .

FITC-conjugated IGF2BP1 antibodies enable researchers to monitor the cellular effects of these therapeutic interventions through high-content imaging, providing visual confirmation of target engagement and downstream effects on protein localization and expression.

What role does IGF2BP1 play in the m6A epitranscriptomic regulatory network?

IGF2BP1 serves as a crucial component in the m6A epitranscriptomic regulatory network with several key functions:

Using FITC-conjugated IGF2BP1 antibodies in combination with antibodies against other m6A regulatory proteins can help visualize the spatial organization and potential interactions within this epitranscriptomic network. Additionally, these tools can be valuable in screening for compounds that disrupt specific interactions within the network.