CD44 Anti-Human, Biotin

What is CD44 and why is it an important research target?

CD44 is a transmembrane glycoprotein that serves as a cell surface receptor involved in cell-cell interactions, cell adhesion, and migration. It exists as multiple isoforms (up to 19 reported) due to alternative RNA splicing. The standard isoform found on lymphocytes, myeloid cells, and erythrocytes is a glycosylated type I transmembrane protein with a molecular weight of approximately 80-95 kDa . Other isoforms containing glycosaminoglycans have molecular weights ranging from 110 to 250 kDa and are widely expressed on both hematopoietic and non-hematopoietic cells .

CD44 contributes to the adhesion of leukocytes to endothelial cells, stromal cells, and the extracellular matrix. It serves as the receptor for hyaluronic acid and plays functional roles in cell migration, lymphocyte homing, and adhesion during hematopoiesis and lymphocyte activation . These diverse functions make CD44 a critical target in research areas including immunology, cancer biology, and stem cell research.

What cell types express CD44 and how can I detect it?

CD44 is expressed on numerous cell types including:

• Leukocytes (lymphocytes, myeloid cells)

• Erythrocytes (weakly on platelets)

• Epithelial cells

• Fibroblasts

• Various stem cell populations

CD44 can be used to identify several specialized cell populations including intestinal crypt stem cells, CD4+ resident memory T cells, and various mesenchymal stromal cells (from Wharton's jelly, amnion, and chorion) .

For detection, biotinylated anti-CD44 antibodies offer versatility across multiple platforms:

• Flow cytometry: Use with streptavidin-conjugated fluorophores (20 μl per test/10^6 cells)

• Immunohistochemistry: Works on both frozen and paraffin sections with appropriate antigen retrieval

• Western blotting: Effective for detecting multiple CD44 isoforms

• Immunoprecipitation: Useful for isolating CD44 and associated proteins

When selecting an antibody, consider specific clones like F10-44-2 (recognizes human/Ma's Night Monkey CD44) or G44-26 (recognizes epitope 1 of human CD44) .

What applications are biotinylated anti-human CD44 antibodies commonly used for?

Biotinylated anti-human CD44 antibodies demonstrate versatility across multiple research applications:

The biotin conjugation provides flexibility, allowing researchers to use various streptavidin-coupled secondary reagents, enhancing detection sensitivity, and enabling multiplexed experimental designs .

How should I optimize flow cytometry protocols using biotinylated anti-CD44 antibodies?

For optimal flow cytometry results with biotinylated anti-CD44 antibodies:

• Sample preparation: Prepare single-cell suspensions with minimal cell aggregation. For adherent cells expressing high CD44 levels, use gentle enzymatic dissociation to preserve the epitope.

• Titration: Although manufacturer recommendations typically suggest 20 μl per test (approximately 10^6 cells) , perform antibody titration to determine optimal concentration for your specific samples.

• Secondary detection: Select appropriate streptavidin-conjugated fluorophores compatible with your cytometer configuration and existing panel. Common conjugates include streptavidin-PE, -APC, or -FITC.

• Controls:

  • Include appropriate isotype controls (Mouse IgG2a-BIOT for F10-44-2 clone or Mouse IgG2b-BIOT for G44-26 clone)

  • Use FMO (Fluorescence Minus One) controls for multicolor panels

  • Include known CD44-positive and CD44-negative cell populations as biological controls

• Buffer considerations: Use buffers containing ≤0.09% sodium azide to prevent internalization of CD44 during staining procedures .

• Analysis: When analyzing data, be aware that CD44 expression can be heterogeneous. Consider both percentage of positive cells and mean fluorescence intensity (MFI) in your analysis.

What are the key considerations for immunohistochemistry applications with biotinylated CD44 antibodies?

When performing immunohistochemistry using biotinylated anti-CD44 antibodies:

• Fixation impact: CD44 epitopes can be sensitive to fixation methods. For formalin-fixed, paraffin-embedded (FFPE) tissues, optimization of antigen retrieval is crucial.

• Endogenous biotin blocking: Tissues may contain endogenous biotin, which can cause background staining. Use an avidin/biotin blocking kit before applying the biotinylated antibody.

• Detection systems: Avidin-biotin complex (ABC) or streptavidin-horseradish peroxidase systems work well with biotinylated antibodies.

• Tissue-specific considerations: CD44 is strongly expressed in lymphoid tissues, epithelial cells, and many tumor types. When staining epithelial tissues, expect strong expression at cell membranes .

• Protocol optimization: For frozen sections, brief fixation in cold acetone (5 minutes) often provides optimal results. For paraffin sections, heat-induced epitope retrieval in citrate buffer (pH 6.0) is typically effective .

• Controls: Include known positive tissues (e.g., lymphoid tissue, skin) and appropriate negative controls (isotype-matched irrelevant antibodies and no primary antibody controls).

How can I effectively use biotinylated anti-CD44 antibodies for western blotting?

For successful western blotting with biotinylated anti-CD44 antibodies:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • Be aware that CD44 is heavily glycosylated and can appear as a smear rather than a sharp band

  • Standard isoform is approximately 80-95 kDa, but other isoforms range from 110 to 250 kDa

• Gel selection:

  • Use 8-10% SDS-PAGE gels for standard isoforms

  • Consider gradient gels (4-15%) for capturing the full range of CD44 isoforms

• Transfer conditions:

  • Use PVDF membranes for better protein retention

  • Optimize transfer time for high molecular weight isoforms

• Detection:

  • After transfer, block with BSA-containing buffer to reduce background

  • Incubate with biotinylated anti-CD44 antibody followed by streptavidin-HRP

  • For enhanced sensitivity, consider chemiluminescent detection systems

• Controls and troubleshooting:

  • Include positive control lysates from cells known to express CD44

  • For complex band patterns, consider deglycosylation experiments to confirm specific isoforms

  • Remember that CD44 can appear at different molecular weights depending on glycosylation and isoform expression

How can I distinguish between different CD44 isoforms using biotinylated antibodies?

Distinguishing between CD44 isoforms requires careful experimental design:

• Antibody selection:

  • Determine if your anti-CD44 antibody recognizes the standard (CD44s) or variant isoforms

  • Clone F10-44-2 recognizes epitopes on the standard region of human CD44

  • For variant isoforms, consider using antibodies targeting specific variant regions

• Western blot analysis:

  • Different isoforms appear at distinct molecular weights:

    • Standard CD44 (CD44s): 80-95 kDa

    • Variant isoforms (CD44v): 110-250 kDa

  • Use gradient gels for better separation

  • Consider enzymatic deglycosylation to distinguish between glycosylation variations and actual isoform differences

• Flow cytometry approach:

  • Combine antibodies recognizing standard and variant regions

  • Analyze co-expression patterns to identify specific isoform populations

  • Use multiparameter analysis to correlate isoform expression with functional markers

• Validation strategies:

  • Complement protein detection with RT-PCR analysis of variant exon inclusion

  • Use cell lines with known isoform expression patterns as controls

  • Consider immunoprecipitation followed by mass spectrometry for definitive isoform identification

Understanding CD44 isoform expression patterns is particularly important in cancer research, where specific variants may correlate with metastatic potential and treatment response .

What strategies can address epitope masking issues when CD44 is heavily glycosylated?

CD44 is subject to extensive post-translational modifications, particularly glycosylation, which can mask epitopes and affect antibody recognition:

• Enzymatic deglycosylation:

  • Treat samples with glycosidases to remove specific glycan structures:

    • PNGase F for N-linked glycans

    • O-glycosidase with neuraminidase for O-linked glycans

  • Compare antibody binding before and after deglycosylation to assess epitope accessibility

• Buffer optimization:

  • Include non-ionic detergents (0.1-0.5% Triton X-100) to improve accessibility to membrane-embedded epitopes

  • Test different pH conditions which can affect glycan conformations

• Alternative fixation methods:

  • Compare methanol, paraformaldehyde, and acetone fixation which can differentially affect glycoprotein epitope accessibility

  • Use mild fixation protocols for flow cytometry to preserve native epitopes

• Multiple antibody approach:

  • Use antibodies recognizing different CD44 epitopes in parallel experiments

  • Compare detection patterns to identify regions affected by glycosylation

These approaches are particularly important when studying CD44 in tissues with high glycosylation levels or when investigating changes in glycosylation patterns during disease progression .

How can biotinylated anti-CD44 antibodies be used to study cancer stem cells and tumor heterogeneity?

CD44 is a marker for cancer stem cells (CSCs) in multiple tumor types. Biotinylated anti-CD44 antibodies offer several advantages for studying CSCs:

• Multiparameter analysis of CSC populations:

  • Design flow cytometry panels using biotinylated anti-CD44 with streptavidin-fluorophores alongside other CSC markers

  • Example panel: CD44-biotin/streptavidin-APC, CD24-FITC, EpCAM-PE, and viability dye

  • This approach allows identification of CSC subpopulations with different phenotypic characteristics

• Tissue microenvironment studies:

  • Use immunohistochemistry to analyze CD44 expression patterns within the tumor microenvironment

  • Implement multiplex IHC to simultaneously detect CD44 and markers of stemness, EMT, or immune cell interactions

  • Study the spatial relationship between CD44+ cells and stromal components

• Functional assays:

  • Sort CD44+ cell populations using biotinylated antibodies and streptavidin-magnetic beads

  • Assess stemness properties through sphere formation assays and limited dilution assays

  • Evaluate treatment resistance profiles of isolated CD44+ subpopulations

• Therapeutic targeting studies:

  • Use biotinylated anti-CD44 to identify and isolate CD44+ cells before and after therapy

  • Assess changes in CD44 isoform expression during treatment resistance development

  • Implement blocking experiments to evaluate the functional importance of CD44 in therapy resistance

The flexibility of biotinylated antibodies allows integration with multiple detection systems, making them valuable tools for comprehensive characterization of heterogeneous tumor populations .

How can I address non-specific binding issues with biotinylated anti-CD44 antibodies?

Non-specific binding can confound results when using biotinylated antibodies. Here are strategies to minimize and control for this issue:

• Optimize blocking conditions:

  • Use 2-5% BSA or normal serum from the same species as the secondary reagent

  • For tissues with high endogenous biotin (liver, kidney, brain), implement specific biotin/avidin blocking steps

  • Use buffers containing <0.1% sodium azide to prevent non-specific binding

• Titrate antibody concentration:

  • Starting with manufacturer's recommended concentration (typically 20 μl per 10^6 cells) , test 2-fold dilutions

  • Determine the optimal concentration that maximizes specific signal while minimizing background

• Include proper controls:

  • Isotype control: Use Mouse IgG2a-BIOT (for F10-44-2 clone) or Mouse IgG2b-BIOT (for G44-26 clone) at the same concentration as the primary antibody

  • FMO controls for multicolor flow cytometry

  • No-primary-antibody controls for IHC/ICC

• Sample preparation considerations:

  • Fresh samples typically show cleaner staining than frozen or fixed samples

  • For fixed samples, optimize fixation time and concentration

  • Remove dead cells using viability dyes as they often bind antibodies non-specifically

• Detection system optimization:

  • When using enzymatic detection systems (HRP/AP), titrate streptavidin-enzyme conjugates

  • For fluorescent detection, select fluorophores with minimal spectral overlap

  • Consider signal amplification methods only after optimizing primary staining conditions

How do I interpret heterogeneous CD44 expression patterns in my samples?

CD44 expression can be heterogeneous within and between samples. Here's how to approach analysis and interpretation:

• Quantitative analysis approaches:

  • For flow cytometry, analyze both percentage of positive cells and mean fluorescence intensity (MFI)

  • Consider using histogram overlays and density plots to visualize population distribution

  • Calculate coefficient of variation (CV) to quantify heterogeneity

• Expression pattern classification:

  • High/homogeneous: Uniform strong expression across the cell population

  • High/heterogeneous: Strong expression with variable intensity

  • Low/heterogeneous: Predominantly low expression with a minority of high-expressing cells

  • Negative: Expression below isotype control threshold

• Biological interpretation frameworks:

  • In cancer samples, heterogeneous CD44 expression may indicate presence of cancer stem cell populations

  • In immune cells, CD44 expression levels often correlate with activation and memory status

  • In epithelial tissues, differential expression may reflect varying stages of differentiation

• Technical vs. biological heterogeneity:

  • Confirm patterns using alternative detection methods (e.g., IF vs. flow cytometry)

  • Perform biological replicates to ensure consistent patterns

  • Use positive control samples with known CD44 expression profiles

Remember that CD44 exists as multiple isoforms resulting from alternative RNA splicing, which can contribute to heterogeneous detection patterns depending on the epitope recognized by your antibody .

What quality control measures should I implement when using biotinylated anti-CD44 antibodies?

To ensure reliable and reproducible results with biotinylated anti-CD44 antibodies:

• Antibody validation:

  • Verify specificity using positive and negative control samples

  • Confirm consistent lot-to-lot performance if using antibodies over extended periods

  • Consider testing multiple clones (e.g., F10-44-2 and G44-26) to confirm staining patterns

• Storage and handling:

  • Store antibodies at recommended temperature (typically 2-8°C)

  • Avoid repeated freeze-thaw cycles

  • Check for precipitates before use and centrifuge if necessary

  • Note lot-specific concentration information

• Experimental controls:

  • Always include appropriate isotype controls (Mouse IgG2a-BIOT or Mouse IgG2b-BIOT)

  • Use biological positive and negative controls

  • For multicolor flow cytometry, implement FMO (Fluorescence Minus One) controls

• Documentation:

  • Record antibody clone, lot number, and concentration used

  • Document all experimental conditions including incubation times, temperatures, and buffer compositions

  • Maintain consistent protocols for longitudinal studies

• Stability monitoring:

  • Test antibody performance periodically on standard samples

  • Be aware that biotin conjugates may show reduced activity over time

  • Monitor background levels which may increase as reagents age

Implementing these quality control measures will help ensure consistent and reliable results across experiments .

How can I optimize CD44 detection in challenging sample types?

Some sample types present specific challenges for CD44 detection:

• Formalin-fixed, paraffin-embedded (FFPE) tissues:

  • Test multiple antigen retrieval methods (citrate pH 6.0, EDTA pH 8.0, enzymatic)

  • Extend retrieval times for older samples

  • Use amplification systems (tyramide signal amplification) for weak signals

  • Consider automated staining platforms for consistent results

• Frozen tissues with high background:

  • Fix sections briefly (2-5 min) in cold acetone rather than formalin

  • Block with 5-10% normal serum + 1% BSA

  • Include 0.1-0.3% Triton X-100 for improved antibody penetration

  • Use Sudan Black B (0.1% in 70% ethanol) to quench autofluorescence

• Single-cell suspensions from solid tissues:

  • Optimize enzymatic digestion to preserve CD44 epitopes

  • Use gentle dissociation methods

  • Include calcium chelators (EDTA) to facilitate cell release while preserving surface proteins

  • Analyze samples within 2-4 hours of preparation

• Highly glycosylated samples:

  • Pre-treat with neuraminidase to remove sialic acid residues

  • Consider periodic acid treatment to modify glycan structures

  • Compare multiple anti-CD44 clones recognizing different epitopes

What complementary techniques can enhance CD44 research beyond antibody-based detection?

While biotinylated anti-CD44 antibodies provide powerful tools for protein detection, complementary approaches can provide additional insights:

• Transcript analysis:

  • RT-PCR for specific CD44 variant detection

  • RNA sequencing to identify alternative splicing patterns

  • Single-cell RNA-seq to correlate CD44 isoform expression with cellular phenotypes

• Functional assays:

  • Hyaluronic acid binding assays to assess CD44 receptor function

  • Migration and invasion assays to evaluate CD44's role in cell motility

  • Competitive binding studies to identify ligand preferences of different isoforms

• Imaging approaches:

  • Super-resolution microscopy to examine CD44 distribution in membrane microdomains

  • Live-cell imaging with fluorescently tagged CD44 to monitor dynamic behaviors

  • FRET (Fluorescence Resonance Energy Transfer) to study CD44 interactions with binding partners

• Genetic manipulation:

  • CRISPR/Cas9 editing to create CD44 knockout models

  • Exon-specific deletion to study variant-specific functions

  • Overexpression systems to test isoform-specific activities

• Mass spectrometry:

  • Glycoproteomics to characterize CD44 post-translational modifications

  • Interactome analysis to identify CD44 binding partners

  • Quantitative proteomics to measure CD44 expression across sample types

Integrating these approaches with antibody-based detection provides a more comprehensive understanding of CD44 biology .

How can CD44 detection methods be applied to translational research questions?

CD44 research has significant translational implications, particularly in cancer and inflammatory diseases:

• Biomarker development:

  • Standardize CD44 detection protocols for clinical samples

  • Correlate CD44 expression patterns with disease progression and treatment response

  • Develop quantitative assays suitable for diagnostic applications

• Therapeutic targeting strategies:

  • Use biotinylated anti-CD44 antibodies to identify and isolate target cell populations

  • Assess effects of CD44-targeted therapies on receptor expression and signaling

  • Monitor changes in CD44 isoform distribution during treatment

• Patient stratification approaches:

  • Implement multiparameter flow cytometry panels including CD44 for immune monitoring

  • Correlate CD44 expression with clinical outcomes

  • Develop predictive models incorporating CD44 status

• Combination therapy research:

  • Investigate interactions between CD44-targeted approaches and standard therapies

  • Use CD44 detection to monitor resistance mechanisms

  • Identify synergistic treatment combinations based on CD44 expression profiles

• Imaging applications:

  • Develop CD44-targeted imaging probes for non-invasive detection

  • Correlate imaging findings with ex vivo CD44 detection

  • Monitor treatment responses using CD44-based imaging biomarkers

These translational applications build upon fundamental research using biotinylated anti-CD44 antibodies, extending their utility from basic science to clinical applications .