Recombinant Human SMAC

Recombinant Human SMAC (Second Mitochondria-derived Activator of Caspases) is used in research applications primarily to study and manipulate apoptotic pathways, particularly by antagonizing inhibitor of apoptosis proteins (IAPs), thereby promoting programmed cell death in various cellular contexts. This makes it a valuable tool for investigating mechanisms of apoptosis, cancer biology, immune modulation, and therapeutic strategies.

Key scientific applications and advantages:

• Apoptosis Research: SMAC promotes apoptosis by binding to and neutralizing IAPs such as XIAP, cIAP1, and cIAP2, which are negative regulators of caspases. This allows researchers to dissect the molecular mechanisms of cell death and survival, especially in cancer and immune cells.

• Cancer Biology and Therapeutics: Recombinant SMAC or SMAC mimetics are widely used to sensitize tumor cells to apoptosis, either alone or in combination with other agents (e.g., TNFα, TRAIL, immune checkpoint inhibitors). This synergy can overcome resistance to cell death in cancer models and is being explored for therapeutic development.

• Immunology and Immune Modulation: SMAC mimetics have been shown to modulate immune responses, such as enhancing macrophage-mediated phagocytosis of tumor cells and rejuvenating exhausted T cells in the tumor microenvironment. They are also used to study the interplay between apoptosis and immune activation.

• HIV and Infectious Disease Research: SMAC mimetics are being investigated for their ability to reverse HIV latency and eliminate latently infected cells, supporting "shock and kill" strategies for HIV cure research.

• Bone Biology: SMAC mimetics can reduce osteoclastogenesis and viability, providing insights into bone degradation mechanisms and potential therapeutic avenues for diseases like multiple myeloma.

Technical advantages of recombinant proteins:

• Consistency and Reproducibility: Recombinant SMAC offers high batch-to-batch consistency, essential for quantitative and mechanistic studies.
• Defined Activity: Recombinant production ensures a well-characterized protein with predictable biological activity, minimizing variability due to impurities or post-translational modifications.
• Engineering Potential: Recombinant SMAC can be modified (e.g., tagged, mutated) to facilitate mechanistic studies, protein-protein interaction assays, or imaging applications.

Best practices:

• Use recombinant SMAC in dose-response and time-course experiments to delineate its effects on apoptosis and related pathways.
• Combine with other agents (e.g., cytokines, chemotherapeutics, immune modulators) to study synergistic effects or resistance mechanisms.
• Validate findings in relevant human cell models, as species-specific differences in SMAC response have been observed.

In summary, Recombinant Human SMAC is a versatile tool for apoptosis, cancer, immunology, and infectious disease research, enabling precise manipulation of cell death pathways and facilitating the development of novel therapeutic strategies.

Yes, recombinant human SMAC can be used as a standard for quantification or calibration in ELISA assays, provided it is of sufficient purity and its concentration is accurately determined. Recombinant proteins are commonly used as standards in quantitative ELISA protocols, including for SMAC/DIABLO.

Key considerations for using recombinant SMAC as an ELISA standard:

• Purity and Quantification: The recombinant SMAC should be highly purified, and its concentration must be precisely measured, typically by absorbance at 280 nm or using a protein assay such as BCA or Bradford.
• Standard Curve Preparation: Prepare serial dilutions of the recombinant SMAC to generate a standard curve covering the expected concentration range in your samples. Typical ranges for SMAC ELISA kits are from low picogram to nanogram per milliliter levels (e.g., 0.12–30 ng/mL, 31.2–2,000 pg/mL).
• Matrix Effects: If your samples are in complex matrices (e.g., plasma, serum), consider diluting the recombinant standard in the same matrix or in assay buffer to minimize matrix effects and ensure comparable binding and detection.
• Validation: Validate the standard curve by spiking known amounts of recombinant SMAC into sample matrices and confirming recovery and linearity.

Protocol best practices:

• Reconstitute lyophilized recombinant SMAC according to manufacturer or laboratory instructions, ensuring complete dissolution.
• Store aliquots at recommended temperatures to maintain protein integrity and avoid freeze-thaw cycles.
• Use the same buffer conditions for standards and samples when possible to reduce variability.

Limitations and caveats:

• Recombinant SMAC may differ slightly from endogenous SMAC in post-translational modifications, which can affect antibody recognition in some assays. Confirm that your ELISA antibodies recognize both recombinant and native SMAC equivalently.
• Large dilution steps can introduce error; perform dilutions carefully and use calibrated pipettes.

Summary Table: Recombinant SMAC as ELISA Standard

In conclusion, recombinant human SMAC is suitable for use as a standard in ELISA quantification, provided you follow best practices for preparation, validation, and assay optimization.

Recombinant Human SMAC (Second Mitochondria-derived Activator of Caspases, also known as DIABLO) has been validated in published research for several key applications, primarily in studies of apoptosis, cancer biology, and immune modulation.

Validated Applications in Published Research:

• Surface Plasmon Resonance (SPR): Used to study protein-protein interactions, particularly with inhibitor of apoptosis proteins (IAPs).
• ELISA Standard: Utilized as a quantitative standard in enzyme-linked immunosorbent assays for detecting SMAC or related apoptotic markers.
• Cell and Tissue Culture: Applied in functional assays to investigate apoptosis induction and IAP inhibition in various cell lines.
• Blocking/Neutralizing Experiments: Used to block or neutralize IAPs in mechanistic studies of apoptosis.
• Protein Array: Incorporated into multiplex protein arrays for high-throughput screening of apoptotic pathways.
• SDS-PAGE: Validated for purity and molecular weight determination in protein characterization studies.

Research Contexts and Experimental Models:

• Cancer Research: Recombinant SMAC and SMAC mimetics have been extensively used to restore sensitivity to apoptotic stimuli (e.g., TNF-α) in cancer cell lines, including melanoma, ovarian carcinoma, and lung cancer xenografts. These studies often combine SMAC with chemotherapeutic agents or TNF-α to assess synergistic effects on cell death and tumor growth inhibition.
• Immunology and Infectious Disease: SMAC mimetics have been validated for reversing HIV latency and promoting immune-mediated clearance of hepatitis B virus-infected hepatocytes. These applications involve both in vitro cell culture and in vivo animal models.
• Macrophage Function: SMAC mimetics have been shown to induce phagocytosis of live tumor cells by human macrophages, highlighting their role in modulating innate immune responses.
• Neurodegenerative Disease Research: SMAC/DIABLO is associated with studies on neurodegeneration, such as Alzheimer's disease, where its role in apoptosis is investigated.

Experimental Techniques:

• Gene Silencing and Knockdown: Recombinant SMAC is used in conjunction with siRNA or CRISPR-Cas9 to study the effects of SMAC depletion on cell proliferation and tumor growth.
• Protein Interaction Studies: SPR and co-immunoprecipitation assays validate SMAC's interaction with survivin and other IAPs.
• Functional Assays: Cell viability, apoptosis induction, and immune cell infiltration are measured following treatment with recombinant SMAC or SMAC mimetics in various models.

Summary Table of Validated Applications

In summary, recombinant human SMAC is a versatile tool validated for use in apoptosis research, cancer therapy studies, immune modulation, and protein interaction assays, with broad utility in both basic and translational biomedical research.

To reconstitute and prepare Recombinant Human SMAC protein for cell culture experiments, follow these general steps, adapting as needed for your specific experimental requirements and the manufacturer's datasheet:

1. Reconstitution of Lyophilized SMAC Protein

• Buffer selection: Most recombinant proteins, including SMAC, can be reconstituted in sterile distilled water or a physiological buffer such as 20 mM HEPES, 0.1 M KCl, pH 7.5, unless otherwise specified by the datasheet.
• Concentration: Reconstitute to a concentration between 0.1–1.0 mg/mL. The exact concentration depends on your downstream application and should be calculated based on the amount of protein and desired final volume.
• Procedure:
  • Allow the vial to equilibrate to room temperature before opening to prevent condensation.
  • Add the appropriate volume of buffer or sterile water directly to the vial.
  • Gently swirl or invert the vial to dissolve the protein. Avoid vigorous shaking or vortexing to prevent denaturation or foaming.
  • Let the solution sit at room temperature for 15–30 minutes with gentle agitation until fully dissolved.

2. Preparation for Cell Culture Use

• Carrier protein: For cell culture, especially at low concentrations, add a carrier protein such as 0.1% BSA or 10% FBS to stabilize the SMAC protein and prevent adsorption to plasticware.
• Sterility: Ensure all solutions are sterile. If necessary, filter the reconstituted protein through a 0.2 μm filter.
• Aliquoting and storage: Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles. Store aliquots at -20°C to -80°C for long-term use. For short-term use (up to one week), store at 2–8°C.
• Additives for stability: For long-term storage, consider adding 5–50% glycerol or trehalose as a stabilizer.

3. Working Solution Preparation

• Dilution: Before adding to cell culture, dilute the reconstituted SMAC protein to the desired working concentration using sterile culture medium or buffer compatible with your cells. Avoid using animal-derived carriers (e.g., BSA, FBS) if performing serum-free or in vivo experiments; use trehalose instead.
• Typical working concentrations: Literature reports using SMAC in the range of 1 nM to 1 μM for functional assays, but optimal concentrations should be empirically determined for your specific cell type and assay.

4. Application

• Add the prepared SMAC protein directly to the cell culture medium at the desired final concentration.
• Include appropriate controls (e.g., vehicle only, positive/negative controls) in your experimental design.

Summary Table: Key Steps for Recombinant Human SMAC Reconstitution

Important: Always consult the specific product datasheet for any unique instructions or formulation details, as buffer composition and reconstitution protocols may vary between suppliers.

If you need a protocol tailored to a specific cell type or application (e.g., apoptosis induction, protein-protein interaction studies), please specify for more detailed guidance.