Background and Biological Significance of ILF3

Interleukin Enhancer Binding Factor 3 (ILF3), also known as NF-AT 90, DRBP76, or TCP110, is a member of the double-stranded RNA-binding protein (dsRBP) family. The protein contains two conserved double-stranded RNA-binding motifs (dsRBMs) that mediate interactions with both RNA and protein partners. ILF3 typically functions as a heterodimer with ILF2 (NF-AT 45) to form the nuclear factor of activated T cells (NF-AT) complex, which binds the antigen recognition response element (ARRE2) of the interleukin-2 (IL-2) promoter and drives T-cell gene expression.

Beyond its role in T-cell activation, ILF3 has been identified as a critical regulator of mRNA stability, acting to protect specific AU-rich element (ARE)-containing transcripts from degradation. ILF3 also participates in microRNA (miRNA) biogenesis by associating with the Drosha processing complex, and it modulates the innate antiviral immune response through interactions with double-stranded RNA intermediates produced during viral replication. These multifaceted roles make ILF3 a protein of significant interest across immunology, virology, and RNA biology research.

Protein Specifications and Quality Attributes

Recombinant Human ILF3 Protein (SKU: rp371650) is produced using recombinant expression systems to ensure batch-to-batch consistency suitable for demanding research applications. Researchers should consult the product datasheet for the confirmed host expression system, tag configuration, molecular weight, purity grade, and endotoxin level specific to this lot.

General quality attributes expected for a research-grade recombinant protein of this class include:

• Purity: Typically ≥85% as determined by SDS-PAGE under reducing conditions; verify exact specification on the current Certificate of Analysis (CoA).
• Endotoxin: Should be below threshold levels appropriate for cell-based functional assays; consult CoA for lot-specific values.
• Formulation: Typically supplied in a carrier protein-containing buffer (e.g., PBS with BSA and glycerol) to maintain stability; confirm with the product datasheet.
• Activity: Functional activity may be confirmed by RNA electrophoretic mobility shift assay (EMSA) or co-immunoprecipitation; consult the datasheet for validated activity data.

Note: All specific numerical values (purity percentage, endotoxin units, exact buffer composition) should be verified against the current product datasheet and CoA provided with each lot.

Reconstitution and Handling Protocol

Proper reconstitution and handling of Recombinant Human ILF3 Protein are essential to preserve biological activity and ensure experimental reproducibility. Follow the steps below as a general guide; always refer to the lot-specific datasheet for any deviations.

1. Equilibration: Allow the vial to equilibrate to room temperature for 10–15 minutes before opening to minimize condensation-related dilution.
2. Reconstitution: If supplied as a lyophilized powder, reconstitute with the volume of sterile deionized water or the buffer specified in the datasheet. Add the reconstitution buffer gently along the inner wall of the vial and allow to dissolve for 10 minutes without vortexing. Gently swirl or flick the vial to achieve complete dissolution.
3. Centrifugation: Briefly centrifuge (e.g., 500–1000 × g for 30 seconds) to collect any material from the vial walls before use.
4. Aliquoting: Prepare single-use working aliquots immediately after reconstitution to prevent activity loss from repeated freeze-thaw cycles. A carrier protein (e.g., 0.1% BSA) may be added to working dilutions to improve stability at low concentrations.
5. Storage of reconstituted protein: Store working aliquots at -80°C. Short-term storage (up to 1 week) at -20°C is acceptable; avoid storage at 4°C for extended periods.

Important: Do not use protein that appears cloudy, has visible precipitate, or has been exposed to multiple freeze-thaw cycles, as this may indicate denaturation and compromised activity.

Experimental Applications and Research Contexts

Recombinant Human ILF3 Protein is suitable for a range of in vitro biochemical and cell-biology research applications:

• Protein–RNA interaction studies (EMSA): ILF3's dsRBMs enable direct binding assays with structured RNA substrates, making it valuable for electrophoretic mobility shift assays to map RNA-binding specificity.
• Western Blot (WB) and SDS-PAGE: Recombinant ILF3 serves as a positive control or standard for antibody validation and quantitative western blotting experiments targeting endogenous ILF3.
• Co-immunoprecipitation (Co-IP) and pull-down assays: Use as bait or prey protein to identify interacting partners within the NF-AT complex or Drosha/DGCR8 microprocessor complex.
• ELISA standard: Can be used to generate standard curves for sandwich ELISA platforms designed to quantify ILF3 in biological samples.
• Transcription factor activity assays: In combination with ILF2, recombinant ILF3 can be used in cell-free transcription systems to study NF-AT complex assembly and DNA-binding activity on the IL-2 ARRE2 element.
• Innate immunity and antiviral research: ILF3's role in dsRNA sensing and interferon pathway regulation makes this recombinant protein suitable for mechanistic studies of antiviral innate immune signaling.

Researchers are encouraged to optimize protein concentrations and buffer conditions empirically for each specific application.

Troubleshooting Common Issues

The following table summarizes common challenges encountered when working with Recombinant Human ILF3 Protein and suggested corrective actions:

• No detectable band on Western blot: Verify antibody compatibility with the tag or epitope used in the recombinant construct. Confirm transfer efficiency with a total protein stain. Ensure protein has not been denatured during handling.
• Protein precipitation or aggregation: Avoid freeze-thaw cycling; add carrier protein (BSA) to diluted aliquots. Work on ice and minimize protein exposure to elevated temperatures. Centrifuge briefly before use to remove aggregates.
• Weak or variable EMSA signal: Optimize salt concentration and pH of the binding buffer, as ILF3-RNA interactions are sensitive to ionic conditions. Ensure RNA substrate is freshly prepared and correctly folded.
• Low activity in functional assays: Confirm that endotoxin levels are within acceptable limits for the cell type used. Check that the protein concentration used falls within the linear range validated in the datasheet.
• Inconsistent results between experiments: Use single-use aliquots and record lot numbers. Standardize reconstitution volumes and buffer composition across experiments.

If problems persist after following these recommendations, contact Aladdin Scientific technical support with your lot number and experimental details for further assistance.