Recombinant Human MIA-2

Recombinant Human MIA-2 is used in research applications primarily because it serves as a tumor suppressor in hepatocellular carcinoma and is a novel acute phase protein predominantly expressed in hepatocytes. Its recombinant form enables precise, reproducible, and consistent experimental results, which are essential for mechanistic studies, biomarker validation, and therapeutic development.

Key scientific reasons to use Recombinant Human MIA-2:

• Tumor Suppression and Cancer Research: MIA-2 functions as a tumor suppressor, particularly in hepatocellular carcinoma, making it valuable for studies investigating cancer mechanisms, tumor progression, and potential therapeutic interventions.

• Acute Phase Protein and Liver Disease Biomarker: MIA-2 is predominantly expressed in hepatocytes and its elevated levels are associated with hepatic disease activity and severity. Recombinant MIA-2 can be used to develop and validate diagnostic assays for liver diseases, and to study acute phase responses in vitro.

• Consistency and Reproducibility: Recombinant proteins provide batch-to-batch consistency, eliminating variability seen with native or animal-derived proteins. This is critical for quantitative assays, mechanistic studies, and high-throughput screening.

• Functional Studies: Recombinant MIA-2 allows for controlled studies of its biological activity, interactions, and signaling pathways, including its role in tumor suppression and acute phase response. It can be used in cell-based assays, protein-protein interaction studies, and pathway analysis.

• Protein Engineering and Structural Biology: Recombinant production enables modifications such as tagging, mutagenesis, or fusion constructs, facilitating purification, detection, and structural studies (e.g., crystallography, cryo-EM).

• Clinical and Translational Research: As a potential biomarker for hepatic disease and cancer, recombinant MIA-2 is essential for developing diagnostic tools, validating clinical assays, and exploring therapeutic applications.

Best practices for using recombinant proteins:

• Validate the protein’s activity and purity using appropriate biochemical and cell-based assays.
• Use standardized protocols for storage, handling, and experimental design to ensure reproducibility.
• Consider the expression system and post-translational modifications relevant to your application.

In summary, Recombinant Human MIA-2 is a scientifically robust tool for research in cancer biology, liver disease, biomarker development, and mechanistic studies due to its defined biological roles and the advantages of recombinant protein technology.

You can use recombinant Human MIA-2 protein as a standard for quantification or calibration in your ELISA assays, provided it is of high purity and its concentration is accurately determined. This approach is standard practice in quantitative ELISA development, especially when purified native protein is unavailable.

Key considerations for using recombinant MIA-2 as an ELISA standard:

• Purity and Quantification: The recombinant MIA-2 should be highly purified (typically >95% by SDS-PAGE and HPLC) and its concentration must be precisely measured, ideally by absorbance at 280 nm or amino acid analysis.
• Standard Curve Preparation: Prepare a serial dilution of the recombinant protein in the same buffer or matrix as your samples to generate a standard curve covering the expected concentration range (e.g., 16–2000 pg/mL for typical MIA-2 ELISA kits).
• Matrix Effects: If your samples are serum, plasma, or other biological fluids, spike the recombinant standard into the same matrix to account for potential matrix effects and ensure accurate quantification.
• Validation: Confirm that the recombinant MIA-2 behaves similarly to endogenous MIA-2 in your assay by assessing parallelism and recovery. This ensures that the assay quantifies both forms equivalently.
• Documentation: Record the lot number, purity, and quantification method for your recombinant standard to ensure reproducibility and traceability.

Best Practices:

• Use freshly prepared or properly stored aliquots of recombinant MIA-2 to avoid degradation.
• Validate the standard curve in each assay run to ensure linearity and reproducibility.
• Whenever possible, calibrate your standard against an international reference material (e.g., NIBSC, WHO) for harmonization.

Summary:
Recombinant Human MIA-2 is suitable as a standard for ELISA quantification if it is highly purified and accurately quantified. Ensure proper validation and matrix matching for reliable calibration.

Recombinant Human MIA-2 has been validated in published research primarily for applications in cancer biology, especially hepatocellular carcinoma (HCC), as well as for studies of its role as a tumor suppressor and potential biomarker for hepatic disease activity and severity.

Key validated applications include:

• In vitro cell-based assays: Recombinant Human MIA-2 has been used to treat HCC cell lines, where it inhibited cell proliferation and invasion, demonstrating its tumor suppressor function. Stable transfection and recombinant protein treatment both resulted in reduced invasive potential and proliferation rate in HCC cells.

• In vivo animal models: MIA-2 re-expressing HCC cells implanted in nude mice showed significantly slower growth and less invasive tumor patterns, validating its role in suppressing tumor growth and invasion in vivo.

• Biomarker studies: Elevated levels of MIA-2 have been investigated as a clinically useful marker for diagnosis and assessment of hepatic disease activity and severity. These studies typically use recombinant protein in immunoassays to quantify MIA-2 levels in biological samples.

• Mechanistic studies: Research has explored the molecular mechanisms by which MIA-2 exerts its tumor suppressor effects, including its regulation by HNF-1 and its impact on tumorigenicity.

• Acute phase protein research: MIA-2 is predominantly expressed in hepatocytes and has been studied as a novel acute phase protein in liver biology.

No published research currently validates recombinant human MIA-2 for clinical diagnostic use or therapeutic applications; its use is restricted to preclinical and basic research settings.

Summary of validated research applications:

• In vitro inhibition of cancer cell proliferation and invasion
• In vivo suppression of tumor growth and invasion in animal models
• Biomarker quantification in hepatic disease research
• Mechanistic studies of tumor suppression and gene regulation
• Acute phase protein studies in liver biology

If you require protocols or specific assay details for any of these applications, please specify the context or experimental system.

Reconstitution Protocol for Recombinant Human MIA-2

Initial Preparation

Begin by centrifuging the vial briefly before opening to concentrate the lyophilized powder at the bottom of the tube. This prevents the powder from adhering to the tube walls or cap, which can occur during transportation and handling.

Reconstitution Buffer Selection and Procedure

Reconstitute the lyophilized MIA-2 protein in sterile distilled water or an aqueous buffer containing 0.1% BSA to achieve a concentration of 0.1–1.0 mg/mL. For example, if you have a 100 µg vial and want to prepare a 1 mg/mL solution, add 100 µL of diluent; for a 0.1 mg/mL solution, add 1 mL. Alternatively, some protocols recommend reconstituting in sterile 18 MΩ·cm H₂O at a minimum concentration of 100 µg/mL.

Allow the vial to reconstitute for 15–30 minutes at room temperature with gentle agitation, avoiding vigorous shaking that can cause foaming and protein denaturation. Gently swirl the vial to ensure complete dissolution of the powder.

Storage Conditions

Short-term Storage

After reconstitution, the protein can be stored at 4°C for 1–7 days depending on your experimental timeline. This storage duration is suitable for experiments with a 5–7 day cycle.

Long-term Storage

For extended storage beyond one week, prepare working aliquots and store at –20°C or below (–80°C preferred). Avoid repeated freeze-thaw cycles, as these can compromise protein activity and stability. The lyophilized protein itself remains stable for at least 2 years when stored desiccated at –20°C.

Optimization for Cell Culture Applications

When performing serum-free culture experiments, ensure the reconstituted protein contains no human or animal-derived proteins such as BSA or fetal bovine serum (FBS). If your protocol requires carrier proteins for stability during dilution, use purified BSA at a final concentration of 10–15 mg/mL or alternative carriers like 0.1% BSA, 10% FBS, or 5% human serum albumin (HAS).

For further dilutions needed in your cell culture experiments, use low endotoxin medium or buffered solutions containing FBS or tissue culture-grade BSA. The optimal working concentration should be determined empirically for your specific application, as different assays and cell types may require different protein concentrations.