FH7 Antibody

What are the different types of FH7 antibodies used in scientific research?

Current scientific literature identifies two distinct antibodies referred to as "FH7":

• Plant Formin-like protein 7 (FH7) antibody: A polyclonal antibody that recognizes Formin-like protein 7 in plants, particularly in rice (Oryza sativa subsp. japonica) and Arabidopsis thaliana .

• FH7 monoclonal antibody for disialosyl Lea: A monoclonal antibody that specifically binds to disialosyl Lea, a carbohydrate antigen that shows differential expression between normal and cancerous tissues .

These antibodies target completely different antigens and are used in distinct research fields, making it essential to specify which FH7 antibody you are referring to in scientific communications.

What is the target antigen for plant FH7 antibody and its biological significance?

The plant FH7 antibody targets Formin-like protein 7 (UniProt ID: Q6K8Z4), a protein expressed in rice (Oryza sativa subsp. japonica) and Arabidopsis thaliana . Formin proteins play crucial roles in cytoskeletal organization, particularly in regulating actin filament assembly and organization. These proteins are involved in fundamental cellular processes including cell division, cell growth, and cellular morphogenesis in plants.

What are the validated applications for plant FH7 antibody?

Based on current validation studies, plant FH7 antibody has been confirmed for the following applications:

For optimal results in Western blot applications, ensure proper sample preparation to maintain protein integrity and use appropriate controls to verify specificity.

What are the recommended storage and handling conditions for plant FH7 antibody?

For optimal stability and performance of plant FH7 antibody:

• Store at -20°C or -80°C for long-term storage

• Avoid repeated freeze-thaw cycles

• When working with the antibody, keep it on ice

• For aliquoting, use sterile tubes and maintain aseptic conditions

• Follow manufacturer's recommendations for reconstitution if supplied in lyophilized form

How can researchers optimize Western blot protocols when using plant FH7 antibody?

When optimizing Western blot protocols with plant FH7 antibody:

• Sample preparation: Use fresh plant tissue and include protease inhibitors in your extraction buffer to prevent degradation of the target protein

• Blocking optimization: Test different blocking agents (BSA vs. non-fat dry milk) to determine which provides the best signal-to-noise ratio

• Antibody dilution: Begin with a 1:1000 dilution and adjust as needed based on signal strength

• Incubation conditions: Overnight incubation at 4°C often yields better results than shorter incubations at room temperature

• Positive control: Include a sample known to express FH7 (e.g., rice seedling tissue) as a positive control

• Negative control: Consider using tissue from FH7 knockout plants or pre-immune serum as negative controls

What is disialosyl Lea and what is its relationship to cancer research?

Disialosyl Lea is a carbohydrate determinant that has an extra sialic acid attached at the C6-position of penultimate GlcNAc in sialyl Lewis a. This glycan structure is preferentially expressed on non-malignant colonic epithelial cells, and its expression significantly decreases during malignant transformation .

The transition from disialosyl Lea-dominant status to sialyl Lewis a-dominant status during malignant transformation has dual functional consequences:

• Loss of normal cell-cell recognition between mucosal epithelial cells and lymphoid cells

• Gain of E-selectin binding activity, which can facilitate hematogenous metastasis

This makes disialosyl Lea an important biomarker for distinguishing between normal and cancerous tissues.

How does the FH7 monoclonal antibody specifically recognize disialosyl Lea?

The FH7 monoclonal antibody specifically binds to disialosyl Lea antigen with high specificity . The ability of the FH7 antibody to detect this sialylated glycan structure can be confirmed through sialidase treatment of tissue sections, which abolishes antibody binding .

This specificity makes FH7 antibody valuable for:

• Studying the distribution of disialosyl Lea in normal and pathological tissues

• Monitoring changes in glycosylation patterns during cancer progression

• Developing diagnostic assays for cancer detection

What experimental methodologies can researchers use to study the transition from disialosyl Lea to sialyl Lewis a during cancer progression?

To investigate this glycosylation transition:

• Comparative immunohistochemistry: Use FH7 antibody (for disialosyl Lea) and N19-9 antibody (for sialyl Lewis a) on serial sections of normal and cancerous tissues

• Gene expression analysis: Measure transcription levels of α2→6 sialyltransferase, which is responsible for disialosyl Lea synthesis and is downregulated in cancer cells

• Epigenetic modification studies: Investigate histone deacetylation and DNA methylation patterns of the α2→6 sialyltransferase gene using techniques like ChIP-seq and bisulfite sequencing

• Cell culture models: Treat cancer cells with butyrate or 5-azacytidine, which can induce disialosyl Lea expression, suggesting epigenetic regulation

• Functional binding assays: Test binding of cells to E-selectin and Siglec-7 before and after transfection with the α2→6 sialyltransferase gene

How can researchers validate the specificity of FH7 antibody binding to disialosyl Lea?

To validate FH7 antibody specificity:

• Sialidase treatment control: Treat one set of samples with sialidase to remove sialic acid residues, which should abolish FH7 binding

• Competitive inhibition: Pre-incubate FH7 antibody with purified disialosyl Lea antigen before applying to samples

• Glycosylation inhibitors: Culture cells with inhibitors of specific glycosylation pathways to confirm the structural requirements for antibody recognition

• Mass spectrometry: Analyze immunoprecipitated material to confirm the presence of disialosyl Lea structure

• Knockout/knockdown studies: Use cells with CRISPR-edited or siRNA-silenced glycosyltransferases involved in disialosyl Lea synthesis

What advanced research applications can benefit from using the FH7 antibody?

The FH7 antibody has potential applications in:

• Cancer diagnostics: Development of assays to detect early changes in glycosylation patterns during carcinogenesis

• Biomarker studies: Investigation of disialosyl Lea as a potential marker for cancer risk or prognosis

• Drug development: Screening compounds that might restore normal glycosylation patterns in cancer cells

• Functional studies: Investigating the role of disialosyl Lea in cell-cell interactions and immune evasion

• Translational research: Creating diagnostic test kits for clinical use to assist in cancer detection and monitoring

How can researchers address cross-reactivity issues when using FH7 antibodies?

Cross-reactivity can be a significant concern when working with antibodies. To address this issue:

For plant FH7 antibody:

• Perform pre-absorption experiments with recombinant FH7 protein to confirm specificity

• Include closely related formin proteins as controls to assess cross-reactivity

• Use tissue from FH7 knockout plants as negative controls

For FH7 monoclonal antibody against disialosyl Lea:

• Test reactivity against related glycan structures (including sialyl Lewis a, Lewis a, and other sialylated glycans)

• Use glycan array technology to comprehensively profile antibody binding specificity

• Perform immunohistochemistry on tissues known to express or lack disialosyl Lea

What are the critical quality control parameters to consider when using FH7 antibodies in research?

Quality control considerations include:

• Antibody validation: Confirm specificity using positive and negative controls appropriate for your experimental system

• Lot-to-lot variation: Test new antibody lots against previous lots to ensure consistent performance

• Signal-to-noise ratio: Optimize blocking and washing conditions to minimize background

• Sample preparation: Ensure consistent processing of samples to allow for valid comparisons

• Quantification standards: Include appropriate standards when performing quantitative analyses

• Technical replicates: Perform multiple technical replicates to assess reproducibility

• Antibody storage: Monitor antibody performance over time to detect any deterioration

What are common causes of false negative results when using FH7 antibodies, and how can they be addressed?

For plant FH7 antibody:

• Protein degradation: Use fresh tissue and include protease inhibitors

• Insufficient protein denaturation: Optimize SDS and heating conditions for Western blot

• Epitope masking: Try different fixation methods for immunohistochemistry

• Low expression levels: Consider enrichment steps or more sensitive detection methods

For FH7 monoclonal antibody against disialosyl Lea:

• Glycan degradation: Minimize neuraminidase activity by using appropriate fixatives and inhibitors

• Glycan synthesis inhibition: Ensure culture conditions don't inadvertently suppress glycosylation

• Epitope accessibility: Test different antigen retrieval methods for fixed tissues

• Competitive inhibition: Check for substances in your sample that might block antibody binding

How can researchers interpret contradictory results between different detection methods when using FH7 antibodies?

When faced with contradictory results:

• Consider epitope accessibility: Different methods expose different epitopes; some may be masked in certain techniques

• Evaluate detection sensitivity: Some methods have higher sensitivity thresholds than others

• Check for interfering substances: Sample components may interfere with one method but not another

• Review protein modifications: Post-translational modifications might affect antibody binding differently across methods

• Validate with orthogonal approaches: Use non-antibody methods (e.g., mass spectrometry, PCR) to confirm findings

• Consider spatial resolution: Some methods detect localized expression that might be diluted in whole-cell measurements

By systematically evaluating these factors, researchers can reconcile contradictory results and gain deeper insights into their experimental system.

Research Applications Table for FH7 Antibodies