Recombinant Human MIA

Using Recombinant Human MIA (Melanoma Inhibitory Activity) protein in research offers several advantages, particularly for studies involving melanoma biology, extracellular matrix interactions, metastasis, and biomarker development.

Key reasons to use recombinant human MIA include:

• Functional and Structural Studies: Recombinant human MIA adopts the native SH3 domain-like fold and retains biological activity comparable to the native protein, making it suitable for mechanistic studies of protein structure, ligand binding, and function.
• Metastasis and Cell Adhesion Research: MIA specifically interacts with fibronectin, competing with integrin binding and thereby promoting cell detachment from the extracellular matrix. This property is central to its role in melanoma cell invasion and metastasis, allowing researchers to model and dissect these processes in vitro and in vivo.
• Biomarker and Diagnostic Research: MIA is a clinically valuable serum marker for metastatic melanoma and has potential as a marker for cartilage damage and rheumatoid arthritis. Recombinant MIA enables the development and validation of immunoassays (e.g., ELISA) for these applications.
• Batch-to-Batch Consistency and Reproducibility: Recombinant production ensures high purity, defined sequence, and consistent biological activity, reducing variability compared to native or serum-derived proteins.
• Versatility in Experimental Design: Recombinant MIA can be used in a variety of assays, including cell migration/invasion assays, protein-protein interaction studies, and as a standard in quantitative assays.
• Ethical and Practical Advantages: Recombinant proteins are produced without the need for animal tissues, supporting ethical research practices and facilitating regulatory compliance.

Additional context:

• Recombinant MIA is often supplied in a lyophilized form for ease of storage and reconstitution, and is typically expressed in systems such as E. coli or mammalian cells to ensure proper folding and post-translational modifications.
• It is strictly for research use and not for therapeutic or diagnostic use in humans.

In summary, recombinant human MIA is a robust tool for investigating melanoma progression, extracellular matrix biology, and biomarker development, offering high reproducibility, functional relevance, and ethical advantages for a wide range of research applications.

Yes, you can use Recombinant Human MIA as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is purified and its concentration is accurately determined.

Several ELISA kits and protocols specifically mention the use of recombinant human MIA as the standard for generating calibration curves. For example:

• The R&D Systems Human MIA Quantikine ELISA Kit uses E. coli-expressed recombinant human MIA as the standard, and the kit is validated for quantifying both recombinant and natural human MIA. The standard curve generated with recombinant MIA is linear and parallel to that obtained with natural MIA, confirming its suitability for calibration.
• The Abcam Human MIA ELISA Kit (ab171340) and other commercial kits also state that they detect both endogenous and recombinant human MIA, and the standards provided are typically recombinant proteins.
• Best practices for ELISA standard preparation recommend using a purified protein or recombinant protein with a known concentration, ideally measured by methods such as HPLC or amino acid analysis, to ensure accurate quantification.

Key considerations:

• Ensure the recombinant MIA is highly purified and free of contaminants.
• Accurately determine the concentration of the recombinant protein.
• Confirm that the recombinant MIA is recognized by the antibodies used in your ELISA assay (i.e., there is no difference in immunoreactivity between recombinant and natural MIA for your specific assay).

In summary, recombinant human MIA is a suitable and commonly used standard for ELISA quantification and calibration, as long as it is properly characterized and compatible with your assay system.

Recombinant Human MIA (Melanoma Inhibitory Activity) protein has been validated for several applications in published research, primarily in the context of cancer biology and extracellular matrix interactions.

Key validated applications include:

• Cell-based functional assays: Recombinant human MIA has been used in Boyden Chamber assays to assess its ability to inhibit the invasive potential of melanoma cell lines, demonstrating that the recombinant protein is biologically active and can reduce melanoma cell invasion in vitro.

• Protein–protein interaction studies: MIA’s interaction with extracellular matrix proteins, especially fibronectin, has been validated using ELISA-based binding assays. These studies showed that recombinant human MIA binds specifically to fibronectin-coated surfaces, but not to other ECM components such as collagen type I or heparan sulfate proteoglycan.

• Structural biology: The three-dimensional structure of recombinant human MIA has been determined using multi-dimensional NMR spectroscopy, confirming its SH3 domain-like fold and providing insights into its functional domains.

• Bioassays for cell attachment: Recombinant MIA has been used to demonstrate its ability to inhibit melanoma cell attachment to fibronectin and laminin, supporting its role in promoting cell detachment and metastasis.

• Serum biomarker studies: While not a direct application of the recombinant protein itself, MIA is widely used as a serum marker for metastatic melanoma and has been proposed as a marker for rheumatoid arthritis and cartilage damage, based on its expression patterns and biological activity.

Summary Table of Validated Applications

Additional Notes:

• Recombinant human MIA is typically validated for use in in vitro research applications and is not approved for clinical diagnostic use.
• Commercial sources often list applications such as ELISA, Western blot, and functional cell-based assays, but the most robust published validations are for the applications above.

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

To reconstitute and prepare Recombinant Human MIA protein for cell culture experiments, follow these steps to ensure protein integrity and biological activity:

1. Centrifuge the Lyophilized Powder

   • Briefly centrifuge the vial before opening to collect all powder at the bottom and minimize loss.

2. Choose the Appropriate Reconstitution Buffer

   • Most recombinant MIA proteins are reconstituted in sterile PBS (phosphate-buffered saline), pH 7.4.
   • If your experiment requires serum-free conditions, ensure the buffer does not contain animal-derived proteins. Trehalose can be used as a stabilizer in such cases.

3. Reconstitution Procedure

   • Add the recommended volume of buffer to achieve the desired concentration (commonly 0.1–1.0 mg/mL).
   • For example, to reconstitute at 400 μg/mL, add 250 μL PBS to 100 μg lyophilized protein.
   • Gently mix by slow pipetting or gentle agitation. Avoid vortexing or vigorous shaking to prevent protein denaturation and foaming.
   • Allow the protein to dissolve at room temperature for 15–30 minutes. If particulates remain, continue gentle mixing at room temperature or 4°C for up to 2–3 hours.

4. Aliquot and Storage

   • Once fully dissolved, aliquot the solution to avoid repeated freeze-thaw cycles.
   • For short-term storage (up to one week), keep at 4°C.
   • For long-term storage, dilute with buffer containing a carrier protein (e.g., 0.1% BSA, 10% FBS, or 5% HAS) or trehalose, then store aliquots at −20°C to −80°C.
   • Avoid multiple freeze-thaw cycles to preserve activity.

5. Preparation for Cell Culture

   • Before use, bring aliquots to room temperature and dilute to the working concentration in cell culture medium.
   • If using serum-free medium, avoid carrier proteins like BSA or FBS; use trehalose or other non-animal stabilizers.
   • Add the protein directly to the culture medium at the desired final concentration.

Additional Notes:

• Always consult the product’s Certificate of Analysis (CoA) or datasheet for specific instructions regarding buffer composition, concentration, and storage conditions.
• If the protein is tagged (e.g., His-tag), confirm compatibility with your assay or cell system.
• For ELISA or bioassay applications, follow the manufacturer’s recommended reconstitution and dilution protocols.

Summary Table: Recombinant Human MIA Protein Reconstitution

This protocol ensures optimal recovery and activity of recombinant human MIA protein for cell culture experiments.