Recombinant Human GEMIN4 Protein: Production & Usage Protocol

Recombinant Human GEMIN4 Protein is a key component of the SMN (Survival of Motor Neuron) complex involved in snRNP biogenesis and pre-mRNA splicing, produced recombinantly to support biochemical and cell biological research applications.

Product Name

Recombinant Human GEMIN4 Protein

SKU

rp372010

Source Organism

Homo sapiens (Human)

Protein Function

Component of the SMN complex; involved in snRNP assembly and RNA processing

Application

Biochemical assays, protein interaction studies, snRNP assembly research

Storage Recommendation

Store at -80°C; avoid repeated freeze-thaw cycles

Background and Biological Significance of GEMIN4

GEMIN4 (Gem-associated protein 4) is an integral component of the SMN (Survival of Motor Neuron) complex, a macromolecular assembly critical for the biogenesis of small nuclear ribonucleoproteins (snRNPs). snRNPs are essential components of the spliceosome, the cellular machinery responsible for pre-mRNA splicing. Within the SMN complex, GEMIN4 interacts with SMN, GEMIN2, GEMIN3, and other Gemin proteins to coordinate the loading of Sm proteins onto small nuclear RNA (snRNA) molecules, a prerequisite for functional spliceosome assembly.

Defects in SMN complex function are associated with Spinal Muscular Atrophy (SMA), a severe neurodegenerative disease. Research on GEMIN4 and its interacting partners provides insight into RNA metabolism, neuronal health, and potential therapeutic targets. Recombinant Human GEMIN4 Protein enables precise in vitro biochemical dissection of these pathways.

Reconstitution and Storage Protocol

Materials Required:

• Recombinant Human GEMIN4 Protein (rp372010)
• Sterile deionized water or appropriate reconstitution buffer (e.g., PBS, pH 7.4)
• Microcentrifuge tubes (low-binding recommended)
• Ice or cooling block

Reconstitution Steps:

1. Briefly centrifuge the vial at low speed (1,000–2,000 × g, 30 seconds) before opening to collect lyophilized material at the bottom.
2. Add the recommended volume of sterile reconstitution buffer (refer to the Certificate of Analysis for the specific lot) slowly along the side of the vial. Do not vortex.
3. Allow the vial to stand on ice for 5–10 minutes, then gently swirl to ensure complete dissolution.
4. Centrifuge briefly to remove any insoluble particulates.
5. Aliquot the reconstituted protein into single-use volumes to minimize freeze-thaw cycles.

Storage Conditions:

• Short-term (up to 1 week): Store at 4°C.
• Long-term: Store at -80°C. Avoid repeated freeze-thaw cycles, as this may compromise protein activity and integrity.
• Addition of a carrier protein (e.g., 0.1% BSA) is recommended for dilute working solutions to prevent adsorption losses.

Experimental Applications and Assay Guidelines

Recombinant Human GEMIN4 Protein (rp372010) is suitable for a range of in vitro biochemical and cell biological applications, including but not limited to:

• Protein–Protein Interaction Studies: Use in co-immunoprecipitation (co-IP), pull-down assays, or surface plasmon resonance (SPR) to map interactions within the SMN complex (e.g., with SMN, GEMIN3, GEMIN5, Sm proteins).
• In Vitro snRNP Assembly Assays: Supplement cell-free systems to reconstitute snRNP assembly and evaluate the functional contribution of GEMIN4.
• Enzyme-Linked Immunosorbent Assay (ELISA): Use as a standard or coating antigen for antibody validation and quantification experiments.
• Western Blot Standards: Serve as a positive control or molecular weight reference for GEMIN4 detection in cell lysates.
• Structural and Biophysical Studies: Suitable as a substrate for cryo-EM, analytical ultracentrifugation, or isothermal titration calorimetry (ITC) experiments aimed at characterizing the SMN complex architecture.

General Assay Tips: Optimize protein concentration empirically for each application. Keep the protein on ice during handling. Pre-clear stock solutions by centrifugation (10,000 × g, 5 min, 4°C) before use in sensitive assays to remove aggregates.

Quality Control and Verification

To ensure experimental reproducibility and data integrity, the following quality control steps are recommended when working with Recombinant Human GEMIN4 Protein:

1. Purity Assessment: Verify protein purity by SDS-PAGE followed by Coomassie staining or silver staining. A single predominant band at the expected molecular weight of GEMIN4 indicates acceptable purity.
2. Identity Confirmation: Confirm protein identity by Western blot using a validated anti-GEMIN4 antibody, or by mass spectrometry peptide mapping.
3. Activity Check: If functional activity is critical, perform a binding assay with a known GEMIN4-interacting partner (e.g., GEMIN3 or SMN protein) to confirm proper folding and interaction competence.
4. Endotoxin Testing: For cell-based assays, verify endotoxin levels using the Limulus Amebocyte Lysate (LAL) assay to ensure results are not confounded by LPS contamination.
5. Concentration Determination: Measure protein concentration using a BCA or Bradford assay, or by absorbance at 280 nm (A280) using the calculated extinction coefficient.

Refer to the product's Certificate of Analysis (CoA) for lot-specific QC data including purity, concentration, and endotoxin levels.

Frequently asked questions

What is the recommended reconstitution buffer for Recombinant Human GEMIN4 Protein?

Sterile PBS (pH 7.4) or the buffer specified on the Certificate of Analysis for the specific lot is generally recommended. Always add buffer slowly along the vial wall and gently swirl rather than vortexing to preserve protein integrity.

How should I store Recombinant Human GEMIN4 Protein to maintain its activity?

For long-term storage, keep the protein at -80°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can lead to protein aggregation and loss of activity. For short-term use within one week, storage at 4°C is acceptable. Adding a carrier protein such as 0.1% BSA to dilute solutions is recommended to minimize adsorption losses.

What applications is Recombinant Human GEMIN4 Protein (rp372010) validated for?

Recombinant Human GEMIN4 Protein is suitable for protein-protein interaction studies (co-IP, pull-down, SPR), in vitro snRNP assembly assays, ELISA as a standard or coating antigen, Western blot as a positive control, and structural or biophysical studies. Please consult the product's Certificate of Analysis for lot-specific validated applications.

What quality control tests are performed on Recombinant Human GEMIN4 Protein?

Lot-specific quality control data, including purity assessed by SDS-PAGE, protein concentration, and endotoxin levels, are documented on the Certificate of Analysis (CoA) accompanying each product. Researchers are encouraged to review the CoA and perform additional identity confirmation by Western blot or mass spectrometry as needed.

What is the biological role of GEMIN4 in the SMN complex?

GEMIN4 is a core subunit of the SMN complex that cooperates with SMN and other Gemin proteins (GEMIN2, GEMIN3, GEMIN5, etc.) to facilitate the assembly of Sm proteins onto small nuclear RNAs (snRNAs), producing mature snRNPs. These snRNPs are essential for pre-mRNA splicing by the spliceosome. Disruption of this pathway is linked to Spinal Muscular Atrophy (SMA).

References

1. Charroux B, Pellizzoni L, Perkinson RA, Yong J, Shevchenko A, Mann M, Dreyfuss G. Gemin4: A novel component of the SMN complex that is found in both gems and nucleoli. J Cell Biol. 2000;148(6):1177–1186.
2. Pellizzoni L. Chaperoning ribonucleoprotein biogenesis in health and disease. EMBO Rep. 2007;8(4):340–345.
3. Wan L, Battle DJ, Yong J, Gubitz AK, Kolb SJ, Wang J, Dreyfuss G. The survival of motor neurons protein determines the capacity for snRNP assembly: biochemical deficiency in spinal muscular atrophy. Mol Cell Biol. 2005;25(13):5543–5551.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.