Background: LBR Biology and Research Significance

The Lamin B Receptor (LBR) is an integral protein of the inner nuclear membrane (INM) belonging to the sterol reductase superfamily. LBR plays a central role in maintaining nuclear architecture by tethering heterochromatin to the nuclear lamina through direct interactions with lamin B, HP1 (heterochromatin protein 1), and histone H3. These interactions are critical for genome organization, gene silencing, and proper nuclear morphology.

Mutations or dysregulation of LBR have been associated with several human diseases, including Pelger–Huët anomaly, hydrops-ectopic calcification-moth-eaten (HEM) skeletal dysplasia, and certain hematological disorders. LBR is also implicated in neutrophil nuclear lobulation, making it a protein of broad interest in developmental biology, nuclear biology, and disease research.

Recombinant Human LBR Protein produced for research use enables mechanistic studies of nuclear envelope assembly, chromatin organization, protein–protein interactions at the INM, and the molecular basis of LBR-associated pathologies.

Reconstitution and Storage Protocol

Materials Required: Sterile deionized water or appropriate reconstitution buffer (e.g., PBS, pH 7.4), microcentrifuge tubes, pipettes, and dry ice or liquid nitrogen for snap-freezing aliquots.

Reconstitution Steps:

1. Briefly centrifuge the vial before opening to collect lyophilized material at the bottom.
2. Add the recommended volume of sterile reconstitution buffer slowly to the side of the vial. Do not vortex; gently swirl or pipette to mix until fully dissolved.
3. Allow the solution to equilibrate at room temperature for 10–15 minutes if needed.
4. Prepare single-use aliquots to minimize freeze-thaw cycles.

Storage Conditions:

• Lyophilized form: Store at -20°C (short-term) or -80°C (long-term, preferred).
• Reconstituted protein: Store at -80°C. Avoid repeated freeze-thaw cycles, which can reduce activity and cause aggregation.
• For working solutions, keep on ice and use within the same day.

Note: Specific reconstitution volume and buffer recommendations may be provided on the product datasheet. Always consult the accompanying documentation for lot-specific guidance.

Application Protocol: Western Blot and Pull-Down Assay

Western Blot (WB) Application:

1. Prepare SDS-PAGE samples by mixing reconstituted LBR protein with 4× loading buffer and heating at 95°C for 5 minutes under reducing conditions.
2. Load appropriate amounts of protein (typically 50–200 ng per lane) alongside a molecular weight marker.
3. Run SDS-PAGE, transfer to PVDF or nitrocellulose membrane, and block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.
4. Incubate with primary anti-LBR antibody overnight at 4°C. Wash 3× with TBST.
5. Incubate with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature. Wash 3× with TBST and develop with ECL substrate.

Pull-Down Assay Application:

1. Couple recombinant LBR protein to GST beads, His-tag resin, or appropriate affinity resin according to the tag present on the recombinant protein.
2. Incubate coupled beads with cell lysate or purified interacting protein candidates (e.g., lamin B, HP1 isoforms) in binding buffer for 2–4 hours at 4°C with gentle rotation.
3. Wash beads 3–5× with binding buffer to remove non-specific interactions.
4. Elute bound proteins with elution buffer (e.g., reduced glutathione for GST, imidazole for His-tag).
5. Analyze eluate by SDS-PAGE and Western blot or mass spectrometry.

Optimize protein concentrations and incubation conditions empirically for your specific experimental system.

Quality Control and Troubleshooting

Quality Control Checks:

• Verify protein identity and apparent molecular weight by SDS-PAGE and Coomassie staining or Western blot using a validated anti-LBR antibody.
• Assess purity by densitometric analysis of SDS-PAGE gels; research-grade recombinant proteins are typically ≥85% pure.
• Confirm protein concentration by BCA or Bradford assay before use.
• For functional assays, include positive and negative controls in each experimental run.

Troubleshooting Common Issues:

Safety, Handling, and Regulatory Notes

Safety Considerations: Recombinant Human LBR Protein is derived from a human gene sequence and should be handled in accordance with institutional biosafety guidelines for recombinant proteins. Standard laboratory personal protective equipment (PPE) — including gloves, lab coat, and eye protection — should be worn at all times when handling this material.

Research Use Only: This product is intended exclusively for in vitro research purposes. It is not approved for use in diagnostic, therapeutic, or clinical applications in humans or animals. Do not administer to humans or animals.

Disposal: Dispose of all protein solutions and contaminated materials in accordance with applicable local, national, and institutional regulations for biological waste.

Documentation: Refer to the product-specific Safety Data Sheet (SDS) and Certificate of Analysis (CoA) provided with each lot for detailed hazard information, purity data, and lot-specific specifications. Contact Aladdin Scientific technical support for additional guidance.