Mouse TNF-alpha Protein: Structure, Function, and Research Applications

Mouse TNF-alpha (Tumor Necrosis Factor-alpha) is a key cytokine involved in immune responses, inflammation, and various physiological processes. Understanding its structure, function, and research applications is crucial for studying the role of TNF-alpha in health and disease. This technical article provides an overview of Mouse TNF-alpha protein, including its structure, signaling pathways, biological functions, and applications in research.

Structure of Mouse TNF-alpha: Mouse TNF-alpha is a homotrimeric protein composed of three identical subunits. Each subunit consists of a transmembrane domain and an extracellular domain. The extracellular domain contains a receptor-binding region and a homologous region responsible for trimerization. The structural features of Mouse TNF-alpha contribute to its biological activity and receptor interactions.

Function and Signaling Pathways: Mouse TNF-alpha primarily acts as a pro-inflammatory cytokine, mediating various immune responses. It plays a crucial role in the regulation of immune cell activation, proliferation, and differentiation. Upon binding to its receptors, TNF-alpha triggers intracellular signaling cascades, such as the NF-κB and MAPK pathways, leading to the activation of downstream target genes involved in inflammation, apoptosis, and immune regulation.

Biological Functions: Mouse TNF-alpha is involved in a wide range of biological processes, including host defense against infections, tissue homeostasis, wound healing, and the development of inflammatory diseases. It regulates the recruitment and activation of immune cells, promotes the production of other cytokines, and influences the balance between inflammation and immune tolerance.

Research Applications: The versatile nature of Mouse TNF-alpha makes it a valuable tool in research applications. It is widely used in various studies, including:

1. Inflammation Research: Mouse TNF-alpha is used to investigate the mechanisms underlying inflammatory responses and the development of inflammatory diseases.
2. Immunology Studies: It is employed to study immune cell activation, differentiation, and the interplay between different immune cell populations.
3. Drug Development: Mouse TNF-alpha serves as a target for therapeutic interventions, and its inhibition has been successful in the treatment of inflammatory conditions such as rheumatoid arthritis.
4. Cancer Research: Mouse TNF-alpha is studied for its role in tumor development, angiogenesis, and the immune response to cancer.

Mouse TNF-alpha is a pivotal cytokine with diverse functions and implications in multiple areas of research. This technical article provides insights into the structure, function, and research applications of Mouse TNF-alpha protein. Understanding the intricacies of TNF-alpha biology contributes to the advancement of knowledge in immunology, inflammation, and therapeutic strategies targeting TNF-alpha-related diseases.

This lab protocol provides a step-by-step guide for the purification of Mouse TNF-alpha protein from a recombinant expression system. The protocol assumes basic knowledge of molecular biology techniques and laboratory equipment.

Materials:

• E. coli expression system containing the Mouse TNF-alpha gene
• LB broth and agar plates supplemented with appropriate antibiotics
• Isopropyl β-D-1-thiogalactopyranoside (IPTG)
• Lysis buffer (e.g., Tris-buffered saline with protease inhibitors)
• Sonication or other cell disruption method
• Centrifuge
• Nickel affinity resin (Ni-NTA agarose or similar)
• Wash buffer (e.g., Tris-buffered saline with imidazole)
• Elution buffer (e.g., Tris-buffered saline with higher concentration of imidazole)
• Dialysis membrane
• SDS-PAGE gel system
• Coomassie blue staining solution
• Western blotting reagents (if desired)

Protocol:

1. Transform the expression vector containing the Mouse TNF-alpha gene into an E. coli expression host strain and plate on LB agar plates containing the appropriate antibiotics. Incubate overnight at the recommended temperature.

2. Pick a single colony and inoculate it into 5 mL of LB broth supplemented with antibiotics. Incubate overnight with shaking at the recommended temperature.

3. Inoculate the overnight culture into a larger volume of LB broth (e.g., 1 L) in a baffled flask and grow to an appropriate cell density (OD600 ~0.6-0.8).

4. Induce protein expression by adding IPTG to a final concentration of 0.5-1 mM. Continue incubation with shaking for an additional 4-6 hours at the recommended temperature.

5. Harvest the cells by centrifugation at a suitable speed (e.g., 4,000 × g for 10 minutes) and discard the supernatant.

6. Resuspend the cell pellet in lysis buffer and disrupt the cells using sonication or another cell disruption method. Ensure complete cell lysis and release of the protein of interest.

7. Centrifuge the lysate at a suitable speed (e.g., 12,000 × g for 15 minutes) to remove cell debris, and collect the supernatant.

8. Load the supernatant onto a column containing the Nickel affinity resin pre-equilibrated with wash buffer. Allow the Mouse TNF-alpha protein to bind to the resin.

9. Wash the column with wash buffer to remove nonspecifically bound proteins and contaminants.

10. Elute the Mouse TNF-alpha protein from the column using elution buffer, which contains a higher concentration of imidazole.

11. Dialyze the eluted protein against an appropriate buffer to remove imidazole and other small molecules.

12. Analyze the purified Mouse TNF-alpha protein using SDS-PAGE gel electrophoresis. Stain the gel with Coomassie blue to visualize the protein bands.

13. Optionally, perform Western blotting using an anti-TNF-alpha antibody to confirm the presence and identity of the Mouse TNF-alpha protein.

14. Quantify the protein concentration using a suitable method such as Bradford assay or BCA assay.

15. Store the purified Mouse TNF-alpha protein in aliquots at -80°C or use it immediately for downstream applications.

This protocol serves as a general guideline. Specific steps and conditions may vary depending on the expression system, purification method, and laboratory equipment available. It is essential to optimize the protocol based on individual requirements and experimental conditions.