Recombinant Human L-Selectin

Recombinant Human L-Selectin is widely used in research because it enables precise, reproducible studies of leukocyte adhesion, migration, and signaling, and serves as a critical tool for assays investigating immune cell trafficking, inflammation, and related pathologies.

Key reasons to use recombinant human L-Selectin in research applications include:

• Functional Studies of Leukocyte Adhesion and Migration: L-Selectin is a key adhesion molecule that mediates the initial "rolling" of leukocytes on endothelial cells, a prerequisite for their migration into lymph nodes and inflamed tissues. Using recombinant protein allows you to dissect these mechanisms in vitro, including binding assays, cell adhesion assays, and migration studies.

• Modeling Immune Cell Trafficking: L-Selectin is essential for lymphocyte homing to secondary lymphoid organs and sites of inflammation. Recombinant protein can be used to model or block these interactions, helping to elucidate the molecular basis of immune surveillance and inflammatory responses.

• Positive Control in Biochemical Assays: Recombinant L-Selectin is commonly used as a positive control in Western blotting, ELISA, immunoprecipitation, and flow cytometry, ensuring assay specificity and sensitivity.

• Ligand Binding and Inhibition Studies: The recombinant protein is valuable for characterizing L-Selectin ligands, screening for inhibitors, or studying the effects of mutations or post-translational modifications on binding affinity and function.

• Therapeutic Target Validation: Since L-Selectin is implicated in chronic inflammatory and autoimmune diseases, recombinant forms are used to validate it as a drug target and to screen for therapeutic candidates that modulate its function.

• Cancer and Infectious Disease Research: L-Selectin-mediated adhesion is relevant in cancer metastasis and pathogen interactions with immune cells, making recombinant protein useful for studying these processes.

• Standardization and Reproducibility: Recombinant proteins provide consistent quality and defined activity, which is critical for reproducible experimental results compared to variable native sources.

In summary, recombinant human L-Selectin is a versatile reagent for dissecting the molecular mechanisms of immune cell trafficking, validating therapeutic targets, and serving as a standard in a variety of immunological and cell biology assays.

You can use recombinant human L-Selectin as a standard for quantification or calibration in your ELISA assays, provided it is of high purity and its concentration is accurately determined. Most commercial ELISA kits for L-Selectin (CD62L) are validated to recognize both natural and recombinant human L-Selectin as standards.

Key considerations:

• Purity and Form: The recombinant protein should be highly purified and, ideally, in a form that matches the epitope recognized by the ELISA antibodies (e.g., correct folding, post-translational modifications if relevant).
• Concentration Determination: Accurately determine the concentration of your recombinant standard, using methods such as absorbance at 280 nm, BCA assay, or HPLC, to ensure reliable calibration.
• 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 for quantification.
• Kit Compatibility: Confirm that your ELISA kit detects both natural and recombinant forms. Most kits for human L-Selectin explicitly state this compatibility.
• Research Use: Note that most recombinant standards and ELISA kits are for research use only and not for diagnostic applications.

Best Practices:

• Use freshly prepared or properly stored aliquots of the recombinant standard to avoid degradation or aggregation.
• Include a control sample of known L-Selectin concentration in each assay to validate your standard curve and quantification accuracy.
• If your recombinant L-Selectin differs in sequence or post-translational modifications from the native protein, verify that the ELISA antibodies still recognize it equivalently.

In summary, recombinant human L-Selectin is widely accepted and commonly used as a standard for ELISA quantification, as long as the above criteria are met and the kit documentation supports its use.

Recombinant Human L-Selectin has been validated for a range of scientific applications in published research, primarily in studies of leukocyte adhesion, migration, and immune cell interactions. The most frequently validated applications include:

• Bioassays: Used to study cell adhesion, leukocyte rolling, and selectin-ligand interactions, including functional assays with cancer cells and immune cells.
• Western Blotting: Employed as a positive control or detection target in protein expression and shedding studies.
• ELISA: Utilized as a standard or control for quantifying L-Selectin or its ligands in biological samples.
• Flow Cytometry: Applied for detection and quantification of L-Selectin expression on cell surfaces, and in functional assays involving leukocyte activation and migration.
• Immunoprecipitation: Used to isolate L-Selectin or its complexes from cell lysates for downstream analysis.
• Binding Assays: Validated for measuring interactions between L-Selectin and its ligands, including glycoproteins and aptamers.
• Cell Selection and Isolation: Recombinant L-Selectin or its ligands have been used in protocols for isolating specific immune cell subsets, such as CD4+ T cells, via aptamer-based or antibody-based methods.
• Functional Blocking/Neutralization: Used in studies to inhibit L-Selectin-mediated processes, such as T-cell homing or leukocyte rolling, often with aptamers or antibodies.
• Immunofluorescence and ICC (Immunocytochemistry): Applied for visualization of L-Selectin localization and expression in cells and tissues.

Published research examples:

• Dynamic biochemical tissue analysis: Used recombinant L-Selectin in bioassays to detect functional selectin ligands on human cancer tissues.
• CRISPR-Cas9 studies: Employed recombinant L-Selectin in flow cytometry to quantify contributions of glycan modifications to leukocyte-endothelium adhesion.
• Cord blood transplantation: Used in bioassays to assess the role of L-Selectin in hematopoietic cell engraftment.
• Western blot detection: Validated for detection of L-Selectin shedding and expression in cell lysates.
• Cell adhesion assays: Immobilized recombinant L-Selectin supports adhesion of cancer cell lines, validating its use in cell adhesion studies.
• Clinical biomarker assays: Used in flow cytometry-based assays to measure L-Selectin shedding as a marker of immune cell activation and response to therapy in inflammatory bowel disease.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and technical protocols, confirming the utility of recombinant human L-Selectin in both basic and translational research.

Reconstitution Protocol

Initial Preparation

Begin by centrifuging the vial before opening to collect any lyophilized powder that may have adhered to the tube walls or cap during transportation. Allow both the vial and reconstitution buffer to equilibrate to room temperature before proceeding, as this ensures optimal protein solubility and activity.

Reconstitution Steps

Add sterile distilled water or phosphate-buffered saline (PBS) to achieve a final concentration of 0.1-0.5 mg/mL, following the specific recommendations provided in your product's Certificate of Analysis. Gently resuspend the protein by light pipetting or gentle agitation for 15-30 minutes at room temperature. Avoid vigorous shaking or vortexing, as this can cause foaming and protein denaturation.

Storage Considerations

Short-term Storage

For experiments requiring use within one week, store the reconstituted protein at 2-8°C in a standard refrigerator. At this temperature, the protein typically remains active for up to seven days, which is suitable for standard cell culture cycles.

Long-term Storage

For extended storage or when the prepared protein cannot be used immediately, dilute the reconstituted solution with a carrier protein-containing solution and store at -20°C to -80°C. Recommended carrier proteins include:

• 0.1% bovine serum albumin (BSA)
• 5% human serum albumin (HSA)
• 10% fetal bovine serum (FBS)
• 5% trehalose (for serum-free applications)

Adding 5-50% glycerol (final concentration) as a cryoprotectant further enhances stability during frozen storage. Aliquot the protein into smaller portions to minimize freeze-thaw cycles, which can compromise protein activity.

Special Considerations for Cell Culture

When performing serum-free culture or in vivo experiments, avoid carrier proteins derived from human or animal sources such as BSA, FBS, or HSA. Instead, use trehalose as the stabilizer, which has been successfully employed to preserve bioactive substances without introducing exogenous proteins.

The reconstituted protein solution should be diluted to working concentrations using carrier protein-containing solutions to prevent protein adhesion to tube walls and maintain activity at lower concentrations.