Recombinant Human Bim L

Recombinant Human Bim L offers several compelling advantages for research applications, particularly in studies focused on apoptosis, protein interactions, and cell death pathways.

Scientific Significance

Bim L (also known as Bod) is a pro-apoptotic protein belonging to the BH3 domain-only subgroup of Bcl-2 family members. This protein plays a critical role in initiating apoptosis and has been identified as an essential regulator in various cellular processes, including thymocyte-negative selection and neuronal apoptosis. Its expression can be induced by nerve growth factor (NGF) and the forkhead transcription factor FKHR-L1, making it particularly relevant for studying neuronal and lymphocyte cell death mechanisms.

Key Research Applications

Protein-Protein Interaction Studies

Recombinant Bim L is invaluable for investigating interactions with other Bcl-2 family members. The protein can form hetero- or homodimers with anti-apoptotic regulators like Bcl-XL and Bcl-2, enabling researchers to study the molecular mechanisms underlying apoptotic regulation. This is particularly useful for examining how Bim L "double-bolt locks" anti-apoptotic proteins, rendering them resistant to displacement by therapeutic compounds.

Drug Development and Screening

Recombinant Bim L serves as an excellent tool for high-throughput screening assays to identify potential drug targets and test the efficacy of BH3-mimetic compounds. By studying how candidate molecules interact with recombinant Bim L in cell-free systems, researchers can evaluate therapeutic potential before advancing to cellular studies.

Structural and Functional Analysis

The recombinant form enables large-scale production for structural biology studies, including X-ray crystallography and other biophysical techniques. This allows researchers to determine protein folding patterns, identify binding sites, and elucidate the molecular mechanisms of apoptotic regulation.

Diagnostic and Validation Assays

Recombinant Bim L can be used as an antigen in diagnostic assays and for validating biomarkers related to apoptosis-dependent diseases, including cancer and neurodegenerative conditions. The high purity and consistency of recombinant preparations improve assay accuracy and reproducibility.

Technical Advantages

Recombinant Bim L provides superior consistency and purity compared to native protein sources, ensuring reproducible experimental results across multiple studies. The protein is typically available with defined molecular weight (approximately 22 kDa for the full-length form) and can be produced in various expression systems optimized for specific research needs. Additionally, recombinant preparations eliminate batch-to-batch variability associated with tissue-derived proteins, making them ideal for rigorous quantitative research.

You can use recombinant human Bim L as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is of high purity, its concentration is accurately determined, and it matches the isoform and epitope recognized by the antibodies in your assay.

Key considerations:

• Purity and Quantification: The recombinant Bim L protein should be highly purified, and its concentration must be precisely known, typically determined by absorbance at 280 nm, BCA, or Bradford assay.
• Isoform and Epitope Compatibility: Ensure that the recombinant Bim L matches the isoform (e.g., BimL, BimEL, BimS) and the specific region recognized by the capture and detection antibodies in your ELISA. Some kits or antibodies may be specific for certain isoforms or epitopes.
• Standard Curve Preparation: Prepare a standard curve using serial dilutions of the recombinant protein in the same buffer or matrix as your samples to minimize matrix effects. The standard curve should cover the expected concentration range of your samples.
• Validation: It is essential to validate that the recombinant Bim L behaves similarly to the endogenous protein in your assay system. This includes confirming parallelism between the standard curve and sample dilution curves, and verifying recovery in spiked samples.
• Formulation: If your recombinant protein is lyophilized, follow reconstitution instructions carefully. If it is in solution, ensure it is stored and handled according to best practices to maintain stability and activity.

Limitations:

• Some commercial ELISA kits are optimized for native protein and may not recognize recombinant forms if there are differences in folding, post-translational modifications, or tags. Always check the kit documentation or perform a pilot experiment to confirm compatibility.
• If the recombinant protein is not full-length or contains tags, ensure these do not interfere with antibody binding.

Summary Table:

In summary, recombinant human Bim L can be used as a standard in ELISA quantification if it is pure, accurately quantified, and validated for your specific assay system. Always perform appropriate controls and validation steps to ensure reliable results.

Recombinant Human Bim L has been validated for several key applications in published research, primarily in studies of apoptosis and cell signaling. The most commonly validated applications include:

• Western Blot (WB): Used to detect Bim L protein expression and assess its molecular weight and post-translational modifications in cell lysates.
• Immunohistochemistry (IHC): Applied to visualize Bim L localization and expression in tissue sections, including cancer and neuronal tissues.
• SDS-PAGE: Utilized for protein purity assessment and molecular weight determination, often as a preparatory step for further biochemical analysis.
• ELISA: Used for quantitative detection of Bim L in biological samples, though less frequently cited than WB and IHC.

Research Areas and Experimental Contexts:

• Cell Biology: Investigation of Bim L’s role in apoptosis, especially its interaction with other BCL-2 family proteins (e.g., BCL2, BCL-XL, MCL1).
• Neuroscience: Studies on neuronal apoptosis and the regulation of Bim L by growth factors and transcription factors.
• Immunology: Analysis of Bim L in lymphocyte apoptosis and thymocyte negative selection.
• Cancer Research: Assessment of Bim L expression in tumor tissues and its involvement in cancer cell death pathways.
• Signal Transduction: Elucidation of pathways leading to Bim L induction and its downstream effects.

Protein-Protein Interaction Studies:

• Recombinant Bim L is frequently used in in vitro binding assays to study interactions with anti-apoptotic proteins (e.g., BCL2, MCL1, BCL-w, Bax), often employing pull-down or co-immunoprecipitation techniques.

Isoform-Specific Functional Studies:

• Bim L is one of several splice variants of Bim, and recombinant forms are used to compare apoptotic potency and functional differences among isoforms.

Summary Table of Validated Applications

Key Insights:

• Bim L is a validated tool for apoptosis research, especially in studies requiring precise detection and quantification of pro-apoptotic proteins.
• Its use spans multiple disciplines, with robust validation in both cell-based and biochemical assays.

If you require protocol details or specific experimental setups for any of these applications, please specify the context or assay type.

To reconstitute and prepare Recombinant Human Bim L protein for cell culture experiments, first consult the product-specific datasheet or Certificate of Analysis for any unique requirements. In the absence of specific instructions, follow these general best practices:

1. Centrifuge the vial: Briefly spin the lyophilized protein vial (e.g., 12,000 × g for 20 seconds) to ensure all powder is at the bottom and not lost when opening.

2. Equilibrate to room temperature: Allow both the vial and the reconstitution buffer to reach room temperature before opening to minimize condensation.

3. Reconstitution buffer:

   • Most recombinant proteins, including Bim L, can be reconstituted in sterile distilled water or 20 mM PBS (phosphate-buffered saline), pH 7.2–7.4.
   • If the datasheet recommends a specific buffer (e.g., with reducing agents or detergents), use that.

4. Add buffer:

   • For a typical concentration, add enough buffer to achieve 0.1–1.0 mg/mL (e.g., for 100 µg protein, add 100–1000 µL buffer).
   • Gently pipette up and down or swirl to dissolve. Avoid vigorous vortexing or foaming, which can denature the protein.

5. Incubation:

   • Let the vial sit at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution.
   • If undissolved material remains, extend incubation up to 2 hours with gentle mixing.

6. Aliquot and storage:

   • For immediate use, keep the reconstituted protein at 2–8 °C and use within one week.
   • For long-term storage, dilute with a buffer containing a carrier protein (e.g., 0.1% BSA or 10% FBS) to prevent adsorption and degradation, then aliquot and store at –20 °C to –80 °C.
   • If using in serum-free or animal experiments, avoid animal-derived carriers and consider using trehalose as a stabilizer.

7. Working solution:

   • Prepare working dilutions in cell culture medium or buffer just before use.
   • If the protein is prone to aggregation or loss, always include a carrier protein in dilutions unless experimental design prohibits it.

Special considerations for Bim L:

• If the protein was expressed in inclusion bodies and requires refolding, ensure all denaturants are removed and the protein is properly refolded before use in cell culture.
• For functional assays (e.g., cytochrome c release), follow the specific protocol for mixing and incubation times as described in relevant literature.

Summary of key steps:

• Centrifuge vial → Add appropriate buffer (sterile water or PBS) → Gently dissolve → Incubate at room temp → Aliquot and store appropriately → Prepare working dilutions with carrier protein if needed.

Always verify with the product datasheet for any unique requirements for Recombinant Human Bim L. If unavailable, the above protocol reflects standard best practices for recombinant protein reconstitution and preparation for cell culture.