Recombinant Human Siglec-2

Recombinant Human Siglec-2 (CD22) is a valuable tool for research applications focused on B cell biology, immune regulation, cancer immunology, and glycan-ligand interactions due to its specific expression on mature B cells and its role as a sialic acid-binding immunomodulatory receptor.

Key scientific reasons to use Recombinant Human Siglec-2 in research:

• B Cell Marker and Functional Studies:
  Siglec-2/CD22 is selectively expressed on mature B cells, making it an ideal marker for studying B cell development, differentiation, and function. Its restricted expression allows for precise targeting in immunological assays and cell sorting.

• Regulation of B Cell Signaling:
  CD22 acts as a coreceptor that modulates B cell receptor (BCR) signaling. It contains immunoreceptor tyrosine-based inhibition motifs (ITIMs) that recruit phosphatases such as SHP-1, leading to negative regulation of BCR activation. Recombinant Siglec-2 enables mechanistic studies of B cell activation, tolerance, and autoimmunity.

• Cancer Immunology and Therapeutic Targeting:
  Siglec-2 is a clinically relevant target in B cell malignancies (e.g., non-Hodgkin lymphoma, acute lymphoblastic leukemia), where it is used for antibody-based therapies and CAR-T cell development. Recombinant Siglec-2 is essential for screening therapeutic antibodies, developing CAR constructs, and evaluating ligand interactions in oncology research.

• Glycan-Ligand Interaction Studies:
  As a sialic acid-binding lectin, Siglec-2 is used to probe glycan structures on cell surfaces, facilitating the identification of physiological ligands and mapping glycosylation patterns in health and disease. Recombinant forms are critical for glycan array screening, proximity labeling, and ligand discovery protocols.

• Assay Development and Screening:
  Recombinant Siglec-2 is used in ELISA, flow cytometry, and cell-based bioassays to quantify ligand binding, receptor activation, and downstream signaling events. It supports high-throughput screening for drug discovery and biomarker validation.

• Immunomodulation and Autoimmunity Research:
  CD22's role in regulating B cell activation makes it a target for studying autoimmune diseases and developing B cell depletion therapies. Recombinant protein enables in vitro and in vivo modeling of immune responses and therapeutic interventions.

Best practices for using Recombinant Human Siglec-2:

• Select the appropriate recombinant format (e.g., Fc chimera, His-tag) for your assay type and detection method.
• Validate bioactivity and purity using SDS-PAGE, SEC-MALS, and functional ligand-binding assays.
• Use recombinant Siglec-2 in combination with glycan arrays, proximity labeling, or antibody screening platforms for comprehensive analysis.

In summary, Recombinant Human Siglec-2 is indispensable for research in B cell immunology, cancer therapy development, glycan biology, and immune regulation due to its unique biochemical properties and clinical relevance.

You can use recombinant human Siglec-2 as a standard for quantification or calibration in ELISA assays, provided it is of high purity, its concentration is accurately known, and it is compatible with your assay format. This is a common practice in quantitative ELISA development and is supported by both general ELISA guidelines and specific product documentation.

Key considerations and supporting details:

• Purity and Quantification: The recombinant protein should be highly purified and its concentration precisely determined, as impurities or inaccurate quantification can compromise the standard curve and assay accuracy.
• Assay Compatibility: The recombinant Siglec-2 must be recognized by the capture and detection antibodies used in your ELISA. Most commercial ELISA kits for Siglec-2/CD22 use recombinant human Siglec-2 as the standard, indicating its suitability for this purpose.
• Standard Curve Preparation: Prepare a serial dilution of the recombinant protein in the same buffer or matrix as your samples to generate a standard curve. This allows for accurate quantification of Siglec-2 in unknown samples by comparison to the standard curve.
• Validation: It is important to validate that the recombinant standard behaves similarly to the native protein in your assay, especially if you are quantifying endogenous Siglec-2 in biological samples. Differences in glycosylation, folding, or tag sequences can sometimes affect antibody recognition or assay performance.
• Documentation: Many ELISA kits and protocols specify the use of recombinant human Siglec-2 as the standard, and some provide detailed instructions for its use in calibration.

Best Practices:

• Use a recombinant standard that matches the sequence and post-translational modifications of the native protein as closely as possible.
• Confirm the linearity and reproducibility of your standard curve in preliminary experiments.
• Store and handle the recombinant standard according to manufacturer or protocol recommendations to maintain stability and activity.

In summary: Recombinant human Siglec-2 is widely used as a standard for ELISA quantification, but ensure it is validated for your specific assay system and that all reagents are compatible for accurate calibration.

Recombinant Human Siglec-2 (CD22) has been validated in published research for a range of applications, primarily in immunology, oncology, and glycan biology. Key validated applications include:

• Bioassays: Used to study ligand binding, B cell signaling, and receptor function in cell-based assays.
• Flow Cytometry: Employed as a detection reagent or target in flow cytometric analysis, particularly in CAR-T cell research targeting CD22 in B-cell malignancies.
• ELISA (Enzyme-Linked Immunosorbent Assay): Utilized as a standard or capture reagent for quantifying binding interactions or protein levels.
• Binding Assays: Applied to characterize interactions with sialic acid-containing ligands and to screen for high-affinity glycan ligands.
• Neutralization Assays: Used to assess the ability of antibodies or ligands to block Siglec-2 function.
• Affinity Purification: Immobilized recombinant Siglec-2 is used to enrich and identify physiological ligands from cell lysates or tissues.
• In Vivo Studies: Validated in animal models to study therapeutic targeting, such as in B cell lymphoma and leukemia models.
• Apoptosis Assays: Used to evaluate the induction of apoptosis in B cells or malignant cells upon engagement with Siglec-2 ligands or antibodies.
• Characterization of Protein-Protein Interactions: Used in structural and biochemical studies to define ligand specificity and binding kinetics.
• CAR-T Cell Research: Recombinant Siglec-2 is used for the development and validation of chimeric antigen receptor (CAR) T cells targeting CD22 in B-cell acute lymphoblastic leukemia (B-ALL) and other malignancies.

Supporting details and examples:

• Bioassays and Flow Cytometry: Recombinant Siglec-2 has been used in bioassays to study B cell receptor (BCR) signaling, ligand binding, and as a target in CAR-T cell validation, including flow cytometric detection of CAR expression and function in engineered T cells.
• ELISA and Binding Assays: Validated as a standard or capture reagent in ELISA and for direct binding assays to characterize glycan and antibody interactions.
• Affinity Purification: Used as an immobilized bait to pull down and identify endogenous ligands from complex biological samples, aiding in the discovery of physiological binding partners.
• In Vivo Applications: Recombinant Siglec-2 has been used in animal models to evaluate therapeutic strategies, such as antibody-drug conjugates or nanoparticle delivery systems targeting B cell malignancies.
• Apoptosis and Functional Assays: Applied to assess the induction of apoptosis in B cells, often in the context of therapeutic antibody or ligand engagement.
• Protein Characterization: Used in structural biology and biophysical assays to determine ligand specificity, affinity, and structural features of Siglec-2 interactions.

Summary Table of Validated Applications

These applications are supported by both primary research articles and product validation data from multiple sources, reflecting the broad utility of recombinant human Siglec-2 in both basic and translational research.

To reconstitute and prepare Recombinant Human Siglec-2 (CD22) protein for cell culture experiments, follow these best practices based on standard protocols and product-specific recommendations:

Reconstitution

1. Centrifuge the vial before opening to ensure the lyophilized powder is at the bottom.
2. Reconstitute the protein in sterile distilled water or PBS, depending on the product specifications. Typical recommended concentrations are:
   • 0.1–0.5 mg/mL for general use (e.g., for cell binding or functional assays).
   • Some suppliers recommend 0.25 mg/mL (e.g., add 400 µL sterile water to a 100 µg vial).
   • For certain products, reconstitution at 0.5 mg/mL or 1 mg/mL may be advised if the amount of lyophilized protein is ≤100 µg.
3. Gently swirl the vial to dissolve the protein. Avoid vortexing or vigorous pipetting to prevent denaturation.

Preparation for Cell Culture

• Filter sterilize the reconstituted protein if necessary (e.g., using a 0.2 µm filter), especially if the formulation does not already include a carrier protein or preservative.
• Aliquot the protein into small volumes to minimize freeze-thaw cycles.
• Add carrier protein (e.g., 0.1% BSA) if recommended by the manufacturer to stabilize the protein during storage.

Storage

• Short-term storage: Store reconstituted protein at 2–8°C for up to one month.
• Long-term storage: Store aliquots at –20°C to –70°C in a manual defrost freezer.
• Avoid repeated freeze-thaw cycles to maintain protein stability and activity.

Use in Cell Culture

• Dilute the reconstituted protein in cell culture medium or buffer as needed for your experiment (e.g., for binding, internalization, or signaling assays).
• Pre-warm the protein solution to 37°C if required for functional assays (e.g., internalization studies).
• Include appropriate controls (e.g., untreated cells, isotype controls, or sialidase-treated cells) to validate experimental results.

Additional Notes

• Always refer to the product-specific datasheet or certificate of analysis for exact reconstitution, storage, and handling instructions, as these may vary by supplier and protein construct (e.g., His-tag, Fc chimera, etc.).
• For functional studies involving Siglec-2, consider pretreating cells with sialidase to modulate ligand availability, as Siglec-2 recognizes sialylated glycoproteins.

By following these guidelines, you can ensure optimal activity and stability of Recombinant Human Siglec-2 protein in your cell culture experiments.