Recombinant Human IFT22 Protein: Handling & Application Protocol

Recombinant Human IFT22 Protein is a research-grade GTPase involved in intraflagellar transport and ciliogenesis, supplied by Aladdin Scientific to support functional, biochemical, and cell-based studies of ciliary assembly and signaling pathways.

Product Name

Recombinant Human IFT22 Protein

SKU

rp375134

Protein Function

Small GTPase involved in intraflagellar transport (IFT) complex B and ciliogenesis

Species Origin

Human (Homo sapiens)

Application

Biochemical assays, protein interaction studies, ciliogenesis research

Storage Recommendation

Avoid repeated freeze-thaw cycles; aliquot upon reconstitution

Overview of IFT22 and Its Biological Significance

IFT22 (Intraflagellar Transport Protein 22), also known as RABL5, is a small Ras-related GTPase that forms an integral component of the intraflagellar transport (IFT) complex B. IFT complex B is essential for anterograde transport within cilia and flagella, moving cargo from the base to the tip of these organelles. Cilia serve critical roles in cellular signaling, fluid movement, and sensory reception, and defects in IFT machinery underlie a class of genetic disorders collectively termed ciliopathies, which include conditions such as Bardet-Biedl syndrome, Joubert syndrome, and primary ciliary dyskinesia.

IFT22 cycles between GDP-bound and GTP-bound states and is believed to regulate the assembly and recycling of IFT particles at the ciliary base. Its interaction with other IFT-B subunits makes it a valuable target for dissecting the molecular mechanisms of ciliary assembly. Recombinant Human IFT22 Protein from Aladdin Scientific enables researchers to investigate these mechanisms in controlled in vitro settings.

Reconstitution and Storage Protocol

Reconstitution: Upon receipt, briefly centrifuge the vial at low speed (e.g., 1,000–2,000 × g for 30 seconds) to collect the lyophilized material at the bottom before opening. Reconstitute the protein in the buffer recommended on the product datasheet (typically sterile phosphate-buffered saline, pH 7.4, or a buffer containing a carrier protein such as 0.1% BSA to enhance stability). Add the reconstitution buffer slowly along the side of the vial and gently swirl — do not vortex vigorously, as this may cause denaturation or aggregation.

Concentration: Reconstitute to the concentration specified on the Certificate of Analysis. For working dilutions, use a compatible assay buffer and prepare fresh dilutions on the day of the experiment.

Aliquoting: Immediately after reconstitution, aliquot the protein into single-use volumes to avoid repeated freeze-thaw cycles, which significantly reduce activity.

Storage: Store the lyophilized protein at −20°C or −80°C as indicated on the product label. Reconstituted protein should be stored at −80°C for long-term use or at 4°C for short-term use (typically up to 1 week). Refer to the Certificate of Analysis for lot-specific stability data.

Note: Specific buffer composition, recommended concentration, and storage temperature should always be confirmed against the product-specific datasheet and Certificate of Analysis provided with each lot.

Experimental Applications and Assay Guidelines

Protein–Protein Interaction Studies: Recombinant Human IFT22 Protein is suitable for pull-down assays and co-immunoprecipitation (co-IP) experiments to characterize binding partners within the IFT-B complex or to identify novel interactors. Ensure that the protein is in its native or near-native conformation by using mild lysis and binding buffers.

GTPase Activity Assays: As a small GTPase, IFT22 can be used in GTP hydrolysis assays (e.g., malachite green colorimetric assays or radiolabeled GTP hydrolysis assays) to evaluate intrinsic GTPase activity or the effect of regulatory proteins such as GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs).

Structural and Biophysical Studies: The recombinant protein may be used as a component in crystallography, cryo-electron microscopy sample preparation, surface plasmon resonance (SPR), or isothermal titration calorimetry (ITC) experiments to determine binding kinetics and thermodynamics with partner molecules.

Cell-Based Reconstitution: Recombinant IFT22 can be introduced into cell-free systems or used to complement IFT22-depleted cellular models to assess functional rescue of ciliogenesis.

Always titrate the protein concentration empirically for each assay type and validate results with appropriate positive and negative controls.

Quality Control and Troubleshooting

Purity Verification: Each lot of Recombinant Human IFT22 Protein undergoes quality control testing. Purity is typically assessed by SDS-PAGE, and biological activity may be confirmed by functional assays. Refer to the Certificate of Analysis for lot-specific purity and activity data.

Aggregation: If the reconstituted protein appears turbid, centrifuge at 10,000–14,000 × g for 5–10 minutes at 4°C and use the supernatant. Aggregation can occur if the protein is reconstituted above the recommended concentration or if the buffer conditions are not optimal.

Loss of Activity: Reduced or absent activity is most commonly caused by repeated freeze-thaw cycles, improper storage temperature, or reconstitution in an incompatible buffer. Always use single-use aliquots and adhere to the recommended storage conditions.

Unexpected Molecular Weight: Minor discrepancies in apparent molecular weight on SDS-PAGE are expected due to post-translational modifications, tags, or anomalous migration of certain proteins. Confirm the expected molecular weight from the product datasheet.

For technical assistance, contact Aladdin Scientific's technical support team with your lot number and Certificate of Analysis.

Frequently asked questions

What is the recommended reconstitution buffer for Recombinant Human IFT22 Protein?

The reconstitution buffer should be confirmed from the product-specific datasheet and Certificate of Analysis provided with your lot. In general, sterile PBS (pH 7.4) with or without a carrier protein such as 0.1% BSA is commonly used for recombinant proteins. Always follow the lot-specific instructions to maintain protein integrity and activity.

How should I store Recombinant Human IFT22 Protein after reconstitution?

After reconstitution, aliquot the protein immediately into single-use volumes and store at −80°C for long-term stability. For short-term use within approximately one week, storage at 4°C is acceptable. Avoid repeated freeze-thaw cycles, as these will progressively reduce protein activity.

What applications is Recombinant Human IFT22 Protein suitable for?

Recombinant Human IFT22 Protein is suitable for a range of in vitro applications including protein–protein interaction studies (pull-down, co-IP), GTPase activity assays, structural and biophysical characterization (SPR, ITC, crystallography), and cell-free reconstitution experiments related to ciliogenesis and intraflagellar transport research.

What is the biological role of IFT22 in the cell?

IFT22 is a small GTPase of the Ras superfamily that functions as a subunit of the intraflagellar transport complex B (IFT-B). It plays a role in regulating IFT particle assembly and recycling at the ciliary base, thereby supporting anterograde ciliary transport and ciliogenesis. Disruption of IFT22 function is associated with ciliopathies.

How can I verify the quality of my lot of Recombinant Human IFT22 Protein?

Each lot is accompanied by a Certificate of Analysis (CoA) detailing purity (typically assessed by SDS-PAGE), concentration, and any functional activity data. Review the CoA upon receipt and contact Aladdin Scientific technical support if the results do not meet the stated specifications.

References

1. Bharat TAM, et al. Structures of actin- and tubulin-based cytoskeletal assemblies by electron cryo-microscopy. Curr Opin Struct Biol. (General reference for structural studies of transport complexes; specific IFT22 structural data should be verified.)
2. Schafer JC, et al. IFTA-2 is a conserved cilia protein involved in pathways regulating longevity and dauer formation in Caenorhabditis elegans. J Cell Sci. 2006;119(Pt 19):4088-4100. (Related to IFT-B subunit function; applicability to human IFT22 should be confirmed.)
3. Taschner M, Lorentzen E. The Intraflagellar Transport Machinery. Cold Spring Harb Perspect Biol. 2016;8(10):a028092. (Review of IFT complex structure and function; verify IFT22-specific details.) doi:10.1101/cshperspect.a028092

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