Recombinant Human Bcl-2-related Protein A1 (aa 1-152)

Recombinant Human BCL2A1 (aa 1-152) is a valuable tool for research applications focused on apoptosis regulation and cell survival mechanisms. Here are the key reasons to incorporate this protein into your research:

Functional Significance

BCL2A1 is an anti-apoptotic member of the BCL-2 family that prevents release of cytochrome c from the mitochondrial intermembrane space into the cytosol. This makes it particularly useful for studying the molecular mechanisms of programmed cell death and survival signaling pathways. The protein functions by binding to the BH3 region of pro-apoptotic BCL-2 family members, effectively neutralizing their pro-death activity.

Research Applications

Apoptosis Assays: The recombinant protein is well-suited for cytochrome c release assays and other mitochondrial permeability studies. You can use it to investigate how anti-apoptotic proteins regulate outer mitochondrial membrane integrity and to measure the kinetics of apoptotic signaling.

Cell Survival Studies: BCL2A1 is naturally expressed in the hematopoietic system where it facilitates survival of selected leukocyte subsets. This makes the recombinant form ideal for studying immune cell biology, lymphocyte development, and inflammation-related research.

Cancer Research: BCL2A1 is overexpressed in various cancer cells, including hematological malignancies and solid tumors. Using recombinant BCL2A1 allows you to model tumor cell survival mechanisms and investigate how cancer cells evade apoptosis, which is critical for developing therapeutic strategies.

Drug Development: The protein serves as an excellent tool for screening small molecule inhibitors targeting BCL2A1, potentially leading to compounds that sensitize tumor cells to apoptosis.

Technical Advantages

The recombinant protein (aa 1-152) lacks the carboxyl-terminal mitochondrial targeting sequence present in natural BCL2A1, yet maintains its ability to neutralize pro-apoptotic BCL-2 family members. This truncated form is advantageous because it provides consistent, reproducible results without complications from variable mitochondrial localization.

The carrier-free formulation is particularly recommended when BSA could interfere with your assay, such as in sensitive binding studies or when protein-protein interactions are being measured.

Yes, you can use Recombinant Human Bcl-2-related Protein A1 (aa 1-152) as a standard for quantification or calibration in ELISA assays, provided it is sufficiently pure and its concentration is accurately determined.

Essential context and supporting details:

• Purity and Quantification: For ELISA standard curves, a purified protein is preferred. Recombinant proteins are commonly used as standards if they are well-characterized and their concentration is reliably measured, typically by absorbance at 280 nm, BCA assay, or HPLC.
• Formulation Considerations: Recombinant Bcl-2-related Protein A1 (aa 1-152) is available in carrier-free and BSA-containing formulations. The carrier-free version is recommended if BSA could interfere with your assay, while the BSA-containing version may offer greater stability for standard preparation.
• Standard Curve Preparation: Prepare serial dilutions of the recombinant protein in an appropriate buffer (often containing BSA for stability) to cover the expected concentration range of your samples. ELISA kits for BCL2A1 typically use standard curves ranging from ~30 pg/mL to 2000 pg/mL.
• Sequence and Tag: The aa 1-152 construct lacks the C-terminal mitochondrial targeting sequence but retains functional domains relevant for antibody recognition in most ELISA formats. If your ELISA antibodies are specific to epitopes within aa 1-152, this recombinant protein is suitable.
• Validation: Ensure the recombinant standard is compatible with your ELISA antibodies (i.e., the antibodies recognize epitopes present in the aa 1-152 region). Some ELISA kits specify the use of full-length or truncated proteins, so confirm your assay’s requirements.

Best practices:

• Accurately determine the concentration of your recombinant protein standard before use.
• Reconstitute and dilute the standard according to best practices for ELISA, using buffers recommended by your assay protocol.
• Validate the standard curve in your assay system to ensure linearity and reproducibility.

In summary, Recombinant Human Bcl-2-related Protein A1 (aa 1-152) is suitable as an ELISA standard if it matches your assay’s antibody specificity and is properly quantified and prepared.

Recombinant Human Bcl-2-related Protein A1 (aa 1-152) has been validated in published research for several key applications, primarily related to its anti-apoptotic function and interactions with other Bcl-2 family proteins.

Validated Applications in Published Research:

• In vitro bioassays:
  The recombinant protein has been used in cytochrome c release assays to assess its ability to inhibit apoptosis by preventing the release of cytochrome c from mitochondria. This is typically performed using isolated mitochondria, recombinant pro-apoptotic proteins (such as cleaved BID), and measuring the effect of Bcl-2-related Protein A1 on cytochrome c release.

• Protein-protein interaction studies:
  The protein has been used to study binding interactions with pro-apoptotic Bcl-2 family members, particularly those containing the BH3 domain. These studies help elucidate the mechanism by which Bcl-2-related Protein A1 neutralizes pro-apoptotic signals.

• In vivo functional studies:
  Recombinant Bcl-2-related Protein A1 has been administered in animal models (e.g., murine models of sepsis) to evaluate its effect on apoptosis and survival outcomes. These studies have demonstrated that extracellular administration of the protein can reduce apoptosis and improve survival in vivo.

• Cancer and transformation models:
  Research has validated the role of Bcl-2-related Protein A1 in supporting cell survival in the context of oncogene-driven transformation (e.g., MYC or ABL kinase-driven blood cancers). These studies often use genetic or protein-based modulation of A1 levels to assess its impact on cell survival and transformation efficiency.

Supporting Details:

• The recombinant protein used in these studies typically lacks the C-terminal mitochondrial targeting sequence but retains its anti-apoptotic activity by binding to the BH3 region of pro-apoptotic proteins.
• Applications are not limited to a single cell type or species; both human and mouse systems have been used for validation.
• The protein is also used to investigate mechanisms of drug resistance in cancer, as its expression is associated with resistance to chemotherapeutic agents in some tumor models.

Summary Table of Validated Applications

Key Points:

• The most common and robustly validated application is the in vitro cytochrome c release assay.
• The protein is also validated for in vivo studies of apoptosis and survival.
• It is a tool for dissecting protein-protein interactions within the Bcl-2 family and for studying mechanisms of oncogenesis and drug resistance.

If you require protocols or more technical details for a specific application, please specify the context.

To reconstitute and prepare Recombinant Human Bcl-2-related Protein A1 (aa 1-152) for cell culture experiments, dissolve the lyophilized protein in deionized sterile water to achieve a final concentration between 0.1–1.0 mg/mL. If the protein is supplied as a liquid, it is typically ready for use but may require dilution in an appropriate buffer.

Detailed protocol and best practices:

• Centrifuge the vial briefly before opening to ensure all powder is at the bottom.
• Add the calculated volume of sterile water to reach your desired concentration (e.g., for 100 µg protein, add 100 µL for 1 mg/mL or 1 mL for 0.1 mg/mL).
• Gently mix by pipetting up and down or vortexing at low speed until fully dissolved.
• For enhanced stability, especially for storage or repeated freeze-thaw cycles, add 5–50% glycerol (final concentration) to the solution. This is particularly important if the protein will be stored at −20 °C or −80 °C.
• Aliquot the solution to avoid repeated freeze-thaw cycles, which can degrade protein activity.
• Store aliquots at −20 °C or −80 °C for long-term use; short-term storage (up to one week) can be at 2–8 °C.
• If the protein is supplied in a buffer containing glycerol, NaCl, or phosphate, it can be used directly or further diluted in cell culture-compatible buffer as needed.

Preparation for cell culture experiments:

• Before adding to cells, ensure the protein solution is sterile. If necessary, filter through a 0.2 µm syringe filter.
• Dilute the reconstituted protein in your cell culture medium or assay buffer to the desired working concentration, typically in the nanomolar to micromolar range depending on experimental design.
• If using in functional assays (e.g., apoptosis inhibition), include appropriate controls and consider adding protease inhibitors and BSA to your buffer immediately prior to use to preserve protein activity.

Additional notes:

• Always consult the product datasheet for any specific buffer requirements or incompatibilities.
• Avoid repeated freeze-thaw cycles to maintain protein integrity.
• If the protein is tagged (e.g., His-tag, GST-tag), ensure compatibility with your downstream applications.

This protocol ensures optimal solubility, stability, and biological activity of recombinant Bcl-2-related Protein A1 for cell culture experiments.