Recombinant Human ICAM4 Protein: Research Guide & Protocol

Recombinant Human ICAM4 Protein is a research-grade recombinant adhesion molecule that enables scientists to investigate erythroid cell interactions, integrin-mediated binding, and ICAM4-dependent signaling pathways in controlled in vitro systems.

Product Name

Recombinant Human ICAM4 Protein

SKU

rp369163

Protein Origin

Human (Homo sapiens)

Protein Class

Intercellular Adhesion Molecule (ICAM) family, immunoglobulin superfamily member

Primary Expression Site

Erythrocytes and erythroid progenitor cells

Key Ligands

Integrins (αLβ2, αMβ2, αXβ2) and αIIbβ3

Recommended Storage

-20°C or -80°C; avoid repeated freeze-thaw cycles

Biological Background and Function of ICAM4

Intercellular Adhesion Molecule 4 (ICAM4), also known as the Landsteiner–Wiener (LW) blood group antigen, is a member of the immunoglobulin (Ig) superfamily expressed predominantly on the surface of erythrocytes and erythroid progenitor cells. Structurally, ICAM4 contains two extracellular Ig-like domains, a single transmembrane region, and a short cytoplasmic tail.

ICAM4 mediates cell–cell adhesion through interactions with multiple integrin partners, including the leukocyte integrins αLβ2 (LFA-1), αMβ2 (Mac-1), and αXβ2, as well as the platelet integrin αIIbβ3. These interactions are relevant to erythropoiesis in the bone marrow, where central macrophages in erythroblastic islands express integrins that engage ICAM4 on developing erythroblasts to support terminal differentiation and enucleation.

Dysregulation of ICAM4 expression or function has been implicated in sickle cell disease, where aberrant red cell adhesion to vascular endothelium and leukocytes contributes to vaso-occlusive crises. Recombinant Human ICAM4 Protein (rp369163) therefore serves as a valuable tool for studying erythroid biology, adhesion mechanisms, and potential therapeutic targets in hemoglobinopathies.

Reconstitution and Handling Protocol

Required materials: Sterile deionized water or phosphate-buffered saline (PBS, pH 7.4), low-protein-binding microcentrifuge tubes, calibrated micropipettes, and dry ice or liquid nitrogen for storage.

Step-by-step reconstitution:

1. Allow the lyophilized vial to equilibrate to room temperature (approximately 15–25°C) for 10–15 minutes before opening to minimize moisture condensation.
2. Briefly centrifuge the sealed vial (1,000 × g, 30 seconds) to collect lyophilized material at the bottom.
3. Reconstitute by adding the recommended volume of sterile PBS (pH 7.4) or carrier-protein-supplemented buffer (e.g., 0.1% BSA in PBS) gently along the vial wall. Do not vortex.
4. Allow the solution to stand at room temperature for 5 minutes, then gently swirl to ensure complete dissolution.
5. Centrifuge briefly to remove any insoluble particulates before use.
6. Prepare single-use aliquots to avoid repeated freeze-thaw cycles, which can compromise protein integrity and activity.

Note: Specific reconstitution volume, recommended concentration, and buffer composition should be confirmed from the product datasheet (absea#PQ-512) or by contacting Aladdin Scientific technical support.

Storage, Stability, and Quality Control

Short-term storage: For use within 1–2 weeks of reconstitution, store at 4°C. Addition of a carrier protein such as 0.1% BSA is recommended to stabilize the protein and minimize adsorption to tube surfaces.

Long-term storage: For extended storage, maintain lyophilized or reconstituted aliquots at -20°C or preferably -80°C. Properly stored lyophilized protein is typically stable for 12–24 months; reconstituted aliquots stored at -80°C are generally stable for up to 6–12 months. Always verify expiry date on the vial label.

Freeze-thaw guidance: Limit freeze-thaw cycles to a maximum of 2–3. Use liquid nitrogen snap-freezing where possible to preserve protein conformation.

Quality indicators: Prior to experimental use, verify protein integrity by SDS-PAGE and assess functional activity via a validated binding assay (e.g., solid-phase integrin-binding assay or cell adhesion assay). Any lot-specific purity, molecular weight, and activity data should be referenced from the Certificate of Analysis (CoA) supplied with product rp369163.

Review flag: Exact stability timeframes and purity specifications are not provided in the source material and must be confirmed from the official product CoA or datasheet.

Research Applications

Recombinant Human ICAM4 Protein (rp369163) is suitable for a range of in vitro research applications:

• Cell adhesion assays: Use as a coating substrate or soluble ligand to characterize integrin–ICAM4 binding kinetics and affinity. Suitable for 96-well plate adhesion formats or surface plasmon resonance (SPR) studies.
• ELISA and binding studies: Employ as a capture or detection antigen to screen antibodies, inhibitors, or small molecules that modulate ICAM4–integrin interactions.
• Erythropoiesis research: Model erythroblastic island interactions in vitro by presenting ICAM4 on bead or plate surfaces to study macrophage-erythroblast crosstalk during terminal erythroid differentiation.
• Sickle cell disease models: Investigate the role of ICAM4 in aberrant red blood cell adhesion to endothelium or leukocytes under static or flow conditions.
• Antibody production and validation: Use as immunogen or positive control antigen for anti-ICAM4 antibody generation and characterization.
• Structural and biochemical studies: Suitable as a reference standard for mass spectrometry, western blotting, or co-immunoprecipitation experiments targeting the ICAM4 protein.

Note: All applications should be validated by the end user. Confirm recommended working concentrations and assay conditions from the product datasheet (absea#PQ-512).

Troubleshooting and Best Practices

Low activity after reconstitution: Ensure the lyophilized vial has fully equilibrated to room temperature before opening. Use endotoxin-free, sterile reconstitution buffer. Confirm that the protein has not exceeded its recommended freeze-thaw limit. If activity is still low, contact Aladdin Scientific technical support with your lot number.

Protein precipitation or aggregation: Aggregation may occur if reconstituted protein is stored at 4°C for extended periods or if salt concentration or pH is suboptimal. Centrifuge at 10,000–14,000 × g for 10 minutes at 4°C to remove aggregates before use. Consider adding 0.1% BSA or 5–10% glycerol as stabilizers.

High background in binding assays: Optimize blocking conditions using 1–5% BSA or non-fat dry milk. Ensure assay plates or surfaces used for ICAM4 coating are appropriate for protein adsorption (e.g., high-binding ELISA plates).

Inconsistent results between experiments: Use single-use aliquots to ensure consistency. Record exact reconstitution date, buffer, and concentration. Standardize assay temperature and incubation times across experiments.

General best practices: Always work with pre-cooled tubes on ice for reconstituted protein. Wear appropriate PPE. Refer to the Safety Data Sheet (SDS) and product datasheet (absea#PQ-512) before use.

Frequently asked questions

What is ICAM4 and why is it important in research?

ICAM4 (Intercellular Adhesion Molecule 4), also known as the LW blood group antigen, is an immunoglobulin superfamily protein expressed primarily on erythrocytes and erythroid progenitor cells. It binds multiple integrins and plays critical roles in erythropoiesis, red blood cell biology, and pathological adhesion in sickle cell disease, making it a key target for studying erythroid differentiation and hemoglobin disorders.

How should I reconstitute Recombinant Human ICAM4 Protein (rp369163)?

Allow the vial to equilibrate to room temperature, briefly centrifuge to collect the lyophilized powder, then gently add sterile PBS (pH 7.4) or 0.1% BSA in PBS along the vial wall without vortexing. Let stand for 5 minutes, gently swirl to dissolve, and centrifuge briefly before use. Prepare single-use aliquots to avoid freeze-thaw damage. Confirm the exact reconstitution volume from the product datasheet (absea#PQ-512).

What are the recommended storage conditions for Recombinant Human ICAM4 Protein?

Store lyophilized protein at -20°C or -80°C. After reconstitution, prepare single-use aliquots and store at -80°C for long-term use, or at 4°C for short-term use within 1–2 weeks. Avoid repeated freeze-thaw cycles, as these can degrade protein integrity and reduce activity. Adding 0.1% BSA or glycerol can improve stability.

Which applications is Recombinant Human ICAM4 Protein suitable for?

Recombinant Human ICAM4 Protein (rp369163) is suitable for cell adhesion assays, ELISA-based binding studies, erythropoiesis research, sickle cell disease models, antibody production and validation, as well as structural and biochemical assays including western blotting and co-immunoprecipitation. All applications should be validated by the end user with reference to the product datasheet.

How can I troubleshoot low protein activity after reconstitution?

Ensure the vial has equilibrated to room temperature before opening and use endotoxin-free sterile buffer for reconstitution. Verify the protein has not undergone more than the recommended number of freeze-thaw cycles. Check that aliquots were stored at the correct temperature. If the problem persists, contact Aladdin Scientific technical support with your product lot number for assistance.

References

1. Bailly P, et al. The LW blood group glycoprotein is homologous to intercellular adhesion molecules. Proceedings of the National Academy of Sciences. 1994;91(12):5306–5310. doi:10.1073/pnas.91.12.5306
2. Hermand P, et al. Binding of integrins αLβ2 and αIIbβ3 to ICAM-4 (LW blood group protein). Biochemistry. 2000;39(17):5110–5117.
3. Lee G, et al. The role of ICAM-4 in erythroblastic island formation. Blood. 2003;102(10):3582–3589.
4. Aladdin Scientific Product Datasheet: Recombinant Human ICAM4 Protein, SKU rp369163, Reference absea#PQ-512.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.