CAM7 Antibody

What is CEACAM7 and what is its biological function?

CEACAM7 (also known as CGM2) is a member of the carcinoembryonic antigen-related cell adhesion molecule family. It functions as a calcium-independent cell adhesion molecule by mediating homophilic binding, which contributes to the maintenance of tissue architecture and stability . This protein is part of the immunoglobulin superfamily and plays important roles in cellular interactions. Understanding the biological function of CEACAM7 is essential for researchers designing experiments to investigate its role in normal physiology and disease states.

What applications are CEACAM7 antibodies typically used for?

CEACAM7 antibodies are commonly used in several laboratory applications:

• Western blotting (WB): For detecting CEACAM7 protein in cell or tissue lysates

• Immunohistochemistry on paraffin-embedded sections (IHC-P): For visualizing CEACAM7 expression in tissue sections

• Immunoprecipitation: For isolating CEACAM7 from complex mixtures

The rabbit polyclonal CEACAM7 antibody described in the literature is suitable for Western blot and IHC-P applications with human samples and recognizes an immunogen corresponding to recombinant fragment protein within human CEACAM7 amino acids 100-250 .

What is the predicted molecular weight of CEACAM7 in Western blotting?

When performing Western blot analysis with CEACAM7 antibodies, researchers should expect to observe a band at approximately 29 kDa, which is the predicted molecular weight of the CEACAM7 protein . This information is crucial for proper interpretation of Western blot results. Variations from this expected size may indicate post-translational modifications, alternative splicing, or potential non-specific binding.

What types of samples can be analyzed with CEACAM7 antibodies?

CEACAM7 antibodies have been validated for use with:

• Human tissue samples (particularly colorectal and stomach cancer tissues)

• Human serum samples

• Recombinant CEACAM7 protein

When using CEACAM7 antibodies for IHC-P, successful staining has been documented in paraffin-embedded human colorectal cancer tissue and formalin-fixed, paraffin-embedded human stomach cancer tissue using DAB (3,3'-diaminobenzidine) as the detection method .

How can I optimize CEACAM7 antibody concentration for different experimental applications?

Optimization of antibody concentration is critical for obtaining specific signals while minimizing background. Based on documented research protocols:

• For Western blotting: Begin with a concentration of 2 μg/mL when detecting recombinant human CEACAM7 protein or CEACAM7 in human serum

• For IHC-P: Start with a concentration of 20 μg/ml for human colorectal and stomach cancer tissues

Optimization process:

• Perform a titration experiment using 2-fold serial dilutions (e.g., 1, 2, 5, 10, 20 μg/mL)

• Include appropriate positive controls (tissues known to express CEACAM7) and negative controls (tissues known not to express CEACAM7 or primary antibody omission)

• Evaluate signal-to-noise ratio at each concentration

• Select the concentration that provides the strongest specific signal with minimal background

What are the key considerations when validating the specificity of a CEACAM7 antibody?

Validating antibody specificity is essential to ensure reliable experimental results. For CEACAM7 antibodies, consider the following approaches:

• Epitope mapping: Confirm the antibody recognizes the expected region (e.g., amino acids 100-250 of human CEACAM7)

• Cross-reactivity testing: Evaluate potential cross-reactivity with other CEACAM family members due to sequence homology

• Knockout/knockdown validation: Test the antibody in CEACAM7 knockout or knockdown samples to confirm signal absence

• Multiple antibody approach: Use antibodies targeting different epitopes of CEACAM7 to corroborate findings

• Recombinant protein controls: Include purified recombinant CEACAM7 as a positive control in Western blots

How can I troubleshoot non-specific binding when using CEACAM7 antibodies?

Non-specific binding can complicate interpretation of results when using CEACAM7 antibodies. Here's a methodological approach to troubleshooting:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, non-fat dry milk, normal serum)

  • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

• Increase washing stringency:

  • Use detergent-containing wash buffers (0.05-0.1% Tween-20 in PBS or TBS)

  • Increase number and duration of washes

• Antibody dilution optimization:

  • Further dilute primary antibody (if background is high)

  • Test shorter incubation times (1-2 hours at room temperature instead of overnight)

• For Western blots specifically:

  • Add 0.1-0.5% SDS to antibody diluent to reduce hydrophobic interactions

  • Pre-adsorb antibody with proteins from non-relevant species

• For IHC-P applications:

  • Perform antigen retrieval optimization (test different buffers and conditions)

  • Include an endogenous peroxidase blocking step

  • Consider using more specific detection systems

What are the advantages and limitations of polyclonal versus monoclonal CEACAM7 antibodies?

Understanding the differences between polyclonal and monoclonal antibodies is important for experimental design:

The rabbit polyclonal CEACAM7 antibody described in the literature recognizes the recombinant fragment protein within human CEACAM7 amino acids 100-250 , offering good sensitivity for applications like Western blotting and IHC-P.

How should I design experiments to study CEACAM7 in cancer tissues?

When investigating CEACAM7 in cancer tissues, consider the following methodological approach:

• Sample selection:

  • Include sufficient number of tumor samples (n≥30 for statistical power)

  • Incorporate matched normal adjacent tissue when possible

  • Consider including colorectal and stomach tissue samples where CEACAM7 expression has been documented

• Experimental controls:

  • Positive control tissues with known CEACAM7 expression

  • Negative control tissues (CEACAM7-negative)

  • Technical controls (primary antibody omission)

• Multiple detection methods:

  • Combine IHC-P (for localization) with Western blotting (for semi-quantification)

  • Consider complementing with mRNA expression analysis (qRT-PCR)

• Quantification approach:

  • Use digital image analysis for objective quantification of IHC staining

  • Establish clear scoring criteria (e.g., H-score, percentage positivity)

  • Ensure blinded scoring by multiple observers

• Clinicopathological correlation:

  • Correlate CEACAM7 expression with patient demographics, tumor stage, grade, and outcome measures

How can I adapt antibody-based methodologies from other immunotherapeutic studies to CEACAM7 research?

Lessons from other antibody-based immunotherapeutic studies can be adapted to CEACAM7 research:

• Antibody engineering approaches:

  • Chimerization techniques: Similar to the CD7 chimeric antibody that demonstrated in vivo function in rheumatoid arthritis patients, CEACAM7 antibodies could be engineered with human constant regions to reduce immunogenicity

  • Antibody-drug conjugate (ADC) development: Following the approach used for anti-CD7 ADCs, where a novel conjugate (J87-Dxd) was generated using a cleavable maleimide-GGFG peptide linker

• Functional assays:

  • Internalization assays: Assess CEACAM7 antibody internalization using flow cytometry, similar to the methodology used for anti-CD7 antibodies

  • Affinity determination: Use Biacore assays to determine affinity constants (kd, ka, and KD) as performed for anti-CD7 antibodies

• Therapeutic potential evaluation:

  • Side-by-side comparison of different antibody formats (monoclonal antibodies, bispecific T-cell engagers, and chimeric antigen receptor T cells) as demonstrated in the Slamf7-targeting model

  • Assessment of potential side effects in preclinical models before advancing to clinical applications

What methodological considerations are important when developing CEACAM7 antibodies for potential therapeutic applications?

While maintaining a research focus (not commercial), these methodological considerations are important:

• Expression system selection:

  • Chinese hamster ovary (CHO) cells are commonly used for therapeutic antibody production, as seen with trastuzumab deruxtecan

  • Murine myeloma cells (Sp2/0) have also been successfully used for therapeutic antibody production

• Antibody format determination:

  • Full-length antibodies: Suitable for applications requiring effector functions

  • Fab fragments: Appropriate when faster clearance and tissue penetration are priorities

  • Antibody-drug conjugates: Consider when enhanced cytotoxicity is desired

• Fc modification considerations:

  • Evaluate the need for Fc modifications to enhance or reduce specific effector functions

  • Consider how these modifications affect half-life and biodistribution

• Methodological testing sequence:

  • In vitro binding assays → internalization studies → functional assays → toxicity screening → in vivo efficacy models

How should I interpret contradictory results when using CEACAM7 antibodies across different experimental platforms?

When faced with discrepancies between platforms (e.g., IHC-P showing positive results but Western blot showing negative results), consider:

• Systematic troubleshooting approach:

  • Verify antibody integrity (avoid freeze-thaw cycles, check expiration)

  • Confirm target protein is not lost during sample preparation

  • Evaluate epitope accessibility in different applications

• Resolution strategies:

  • Use multiple antibodies targeting different CEACAM7 epitopes

  • Complement antibody-based methods with nucleic acid detection (RT-PCR, RNA-seq)

  • Consider post-translational modifications that might affect epitope recognition

• Data interpretation framework:

  • Western blot detects denatured protein while IHC may detect native conformation

  • Some epitopes may be masked in certain applications

  • Expression levels may be below detection threshold in some assays

What statistical approaches are most appropriate for analyzing CEACAM7 expression data in clinical samples?

When analyzing CEACAM7 expression data from clinical samples:

• Quantitative analysis of IHC data:

  • H-score calculation (staining intensity × percentage of positive cells)

  • Automated image analysis for objective quantification

  • Receiver operating characteristic (ROC) curve analysis to determine optimal cutoff values

• Statistical tests for group comparisons:

  • For normally distributed data: t-test (two groups) or ANOVA (multiple groups)

  • For non-normally distributed data: Mann-Whitney U test (two groups) or Kruskal-Wallis test (multiple groups)

  • For paired samples: Paired t-test or Wilcoxon signed-rank test

• Survival analysis approaches:

  • Kaplan-Meier curves with log-rank test for comparing survival between groups

  • Cox proportional hazards regression for multivariate analysis

  • Competing risk analysis when multiple outcome events are possible

• Sample size considerations:

  • Power calculations based on expected effect size

  • Consideration of multiple testing correction (e.g., Bonferroni, False Discovery Rate)

How might CEACAM7 antibodies be incorporated into multi-parameter immunophenotyping approaches?

Advanced multi-parameter approaches for CEACAM7 investigation:

• Multiplex immunofluorescence methodology:

  • Combine CEACAM7 antibody with markers for cell types, activation states, and other CEACAM family members

  • Use spectral unmixing to resolve overlapping fluorophores

  • Implement tyramide signal amplification for enhanced sensitivity

• Mass cytometry (CyTOF) integration:

  • Label CEACAM7 antibody with rare earth metals

  • Combine with dozens of other markers for high-dimensional analysis

  • Apply dimensionality reduction techniques (tSNE, UMAP) for data visualization

• Spatial transcriptomics correlation:

  • Correlate CEACAM7 protein expression with spatial gene expression data

  • Map CEACAM7-expressing cells within tissue microenvironments

• Single-cell analysis considerations:

  • Combine single-cell RNA-seq with antibody-based protein detection (CITE-seq)

  • Integrate CEACAM7 antibodies into single-cell proteomics workflows

What emerging technologies might enhance the specificity and utility of CEACAM7 antibodies in research applications?

Emerging methodological approaches for CEACAM7 antibody research:

• Recombinant antibody technologies:

  • Phage display selection for identifying high-affinity CEACAM7-binding antibody fragments

  • Yeast display for affinity maturation of existing CEACAM7 antibodies

  • Synthetic antibody libraries to generate CEACAM7 antibodies with novel properties

• Proximity-based detection methods:

  • Proximity ligation assay (PLA) for detecting CEACAM7 interactions with binding partners

  • Enzyme complementation assays for studying CEACAM7 dynamics

• Optogenetic and chemogenetic approaches:

  • Photo-activatable antibody fragments for spatiotemporal control of CEACAM7 targeting

  • Antibody-based chemical-induced dimerization systems

• Nanobody development:

  • Single-domain antibodies against CEACAM7 for improved tissue penetration

  • Site-specific labeling strategies for nanobody-based imaging

How should researchers integrate CEACAM7 antibody findings with other molecular and cellular data?

Integrative approaches for CEACAM7 research:

• Multi-omics integration framework:

  • Correlate protein expression data from CEACAM7 antibody studies with transcriptomics, epigenomics, and proteomics data

  • Implement computational approaches (network analysis, pathway enrichment) to contextualize CEACAM7 in biological systems

  • Consider temporal dynamics when integrating data from different platforms

• Functional validation methodology:

  • Use CEACAM7 antibodies in conjunction with genetic manipulation (CRISPR/Cas9, RNAi)

  • Correlate antibody-based detection with functional assays (adhesion, migration, proliferation)

  • Develop conditional systems for temporal control of CEACAM7 function

• Translational research considerations:

  • Correlate CEACAM7 expression with clinical parameters and outcomes

  • Evaluate potential as diagnostic, prognostic, or predictive biomarker

  • Assess therapeutic targeting potential based on expression patterns