Recombinant Human IFT88 Protein: Handling & Application Protocol

Recombinant Human IFT88 Protein is a critical tool for investigating intraflagellar transport, primary cilia assembly, and related signaling pathways in mammalian cell biology research.

Product Name

Recombinant Human IFT88 Protein

SKU

rp372025

Gene / Protein

IFT88 (Intraflagellar Transport Protein 88), also known as TG737 and Polaris

Protein Complex Membership

IFT Complex B component

Biological Function

Essential for primary cilia assembly, maintenance, and Hedgehog signaling transduction

Recommended Storage

Store lyophilized or reconstituted protein at -20°C or -80°C; avoid repeated freeze-thaw cycles

Supplier Reference

absea #PP-11982

Background and Biological Significance of IFT88

Intraflagellar Transport Protein 88 (IFT88), encoded by the IFT88 gene and also known as TG737 or Polaris, is a highly conserved tetratricopeptide repeat (TPR)-containing protein that serves as an essential structural component of the IFT Complex B. Intraflagellar transport (IFT) is the bidirectional movement of multi-protein complexes along the axonemal microtubules of cilia and flagella, driven by kinesin-2 (anterograde) and cytoplasmic dynein-2 (retrograde) motor proteins.

IFT88 plays an indispensable role in the assembly and maintenance of primary cilia — non-motile, antenna-like organelles present on the surface of virtually all vertebrate cell types. Primary cilia serve as signaling hubs for key developmental pathways, most notably the Hedgehog (Hh) signaling pathway, as well as platelet-derived growth factor receptor (PDGFR) and Wnt pathways. Loss-of-function mutations or knockdown of IFT88 results in failure of ciliogenesis, leading to a spectrum of developmental defects and ciliopathies including polycystic kidney disease (PKD), Bardet-Biedl syndrome, and Joubert syndrome.

Recombinant Human IFT88 Protein enables researchers to conduct in vitro biochemical assays, protein-protein interaction studies, and functional reconstitution experiments to dissect the molecular mechanisms underlying IFT and ciliary biology.

Reagent Preparation and Reconstitution

Materials Required:

• Recombinant Human IFT88 Protein (rp372025)
• Sterile deionized water or appropriate reconstitution buffer (e.g., PBS, pH 7.4, with 0.1% BSA as carrier protein)
• Low-protein-binding microcentrifuge tubes
• Ice bucket and dry ice for temperature-controlled handling

Reconstitution Procedure:

1. Allow the lyophilized vial to equilibrate to room temperature for 10–15 minutes before opening to minimize moisture condensation.
2. Briefly centrifuge the vial (1,000 × g, 30 seconds) to collect lyophilized material at the bottom.
3. Reconstitute the protein by adding the recommended volume of sterile reconstitution buffer slowly and gently along the vial wall. Do not vortex.
4. Allow the vial to stand at room temperature for 5–10 minutes, then gently swirl to ensure complete dissolution.
5. Centrifuge at low speed (500–1,000 × g, 2 minutes) to remove any insoluble particles.
6. Aliquot into single-use volumes in low-protein-binding tubes to avoid repeated freeze-thaw cycles.

Note: The exact reconstitution volume and buffer formulation should be verified against the Certificate of Analysis (CoA) supplied with each lot. Specific concentration and buffer details for rp372025 should be confirmed with the product datasheet.

Storage Conditions and Stability

Proper storage is essential to maintain the biological activity and structural integrity of Recombinant Human IFT88 Protein:

• Lyophilized form: Store at -20°C, protected from moisture and light. Lyophilized protein is generally stable for up to 12 months from the date of manufacture when stored correctly.
• Reconstituted protein: For short-term storage (up to 1 week), keep at 2–8°C. For long-term storage, aliquot and store at -80°C. Avoid repeated freeze-thaw cycles, as this can lead to protein aggregation and loss of activity.
• Working solutions: Prepare fresh dilutions in assay buffer on the day of the experiment where possible. If a carrier protein (e.g., 0.1% BSA) is not included in the reconstitution buffer, consider adding it to stabilize the protein at low concentrations.
• Freeze-thaw cycles: Limit to no more than 3 freeze-thaw cycles. Each cycle can reduce protein activity; single-use aliquots are strongly recommended.

Always refer to the lot-specific Certificate of Analysis and product datasheet for confirmed stability data for rp372025.

Experimental Applications

Recombinant Human IFT88 Protein is suitable for a range of in vitro and cell-free experimental applications in cilia biology and intraflagellar transport research:

• Protein–Protein Interaction Studies: IFT88 interacts with multiple IFT Complex B partners, including IFT52, IFT70, and IFT57. Pull-down assays, co-immunoprecipitation setups, and surface plasmon resonance (SPR) experiments can be used to characterize these interactions and identify novel binding partners.
• ELISA and Immunoassay Development: Recombinant IFT88 can serve as a standard or capture/detection antigen in sandwich ELISA formats for quantification of IFT88 in biological samples.
• Antibody Validation: Use as a positive control antigen to confirm specificity and sensitivity of anti-IFT88 antibodies in Western blot and immunoprecipitation.
• In Vitro Functional Assays: Reconstitution of partial IFT complex assemblies to study stoichiometry and cooperative binding within the IFT-B sub-complex.
• Structural Biology: Suitable as input material for cryo-electron microscopy (cryo-EM) or X-ray crystallography studies aimed at resolving IFT88 domain architecture or complex structures.
• Hedgehog Pathway Research: IFT88 is mechanistically linked to Smoothened (SMO) and GLI transcription factor trafficking within primary cilia; recombinant IFT88 can be used in in vitro assays probing these interactions.

Quality Control and Troubleshooting

Quality Control Considerations:

Each lot of Recombinant Human IFT88 Protein (rp372025) is manufactured under controlled conditions. Researchers should review the Certificate of Analysis (CoA) for lot-specific information on purity (typically assessed by SDS-PAGE), identity confirmation, and endotoxin levels before use in sensitive cell-based assays.

Common Issues and Troubleshooting:

Issue	Likely Cause	Recommended Action
Protein precipitation after reconstitution	Incorrect buffer, vortexing, or temperature shock	Use recommended buffer; reconstitute gently; equilibrate to room temperature first
Low signal in ELISA or Western blot	Protein degradation or inadequate loading	Verify storage conditions; quantify protein before use (BCA or Bradford assay)
Loss of binding activity in pull-down	Repeated freeze-thaw or protein aggregation	Use fresh aliquots; add protease inhibitors; verify protein integrity by SDS-PAGE
High background in immunoassays	Endotoxin contamination or non-specific binding	Check endotoxin level on CoA; optimize blocking conditions

For additional technical support, contact Aladdin Scientific customer service with your lot number and experimental details.

Frequently asked questions

What is the function of IFT88 in primary cilia biology?

IFT88 is a core component of IFT Complex B that is essential for the anterograde transport of cargo along ciliary axonemes. It is required for the assembly and maintenance of primary cilia, and its loss leads to ciliogenesis failure, disruption of Hedgehog signaling, and ciliopathies such as polycystic kidney disease.

How should Recombinant Human IFT88 Protein (rp372025) be stored to maintain activity?

Lyophilized protein should be stored at -20°C away from moisture and light. After reconstitution, aliquot the protein into single-use volumes and store at -80°C for long-term use or at 2–8°C for up to one week. Avoid repeated freeze-thaw cycles, as these can lead to aggregation and reduced biological activity.

What experimental applications is Recombinant Human IFT88 Protein suitable for?

Recombinant Human IFT88 Protein is suitable for protein-protein interaction studies (pull-down, SPR), ELISA standard and antigen development, antibody validation by Western blot and immunoprecipitation, in vitro IFT complex reconstitution assays, and structural biology applications such as cryo-EM. It is also used in research on Hedgehog pathway components that traffic through primary cilia.

How do I reconstitute the lyophilized IFT88 protein correctly?

Allow the sealed vial to equilibrate to room temperature for 10–15 minutes, then briefly centrifuge to collect all material. Add sterile reconstitution buffer (e.g., PBS pH 7.4 with 0.1% BSA) slowly along the vial wall without vortexing. Let stand for 5–10 minutes, gently swirl, then centrifuge at low speed to remove any particulates. Always confirm the exact recommended volume and buffer on the lot-specific CoA.

Is Recombinant Human IFT88 Protein suitable for use in cell-based assays?

It may be used in cell-based contexts depending on delivery method and experimental design; however, endotoxin levels must be verified from the lot CoA before use in cell-based assays, as endotoxin contamination can confound results, particularly in signaling pathway studies. Contact Aladdin Scientific for lot-specific endotoxin data.

References

1. Rosenbaum JL, Witman GB. Intraflagellar transport. Nat Rev Mol Cell Biol. 2002;3(11):813-825. doi:10.1038/nrm952
2. Pazour GJ, Dickert BL, Vucica Y, et al. Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella. J Cell Biol. 2000;151(3):709-718. doi:10.1083/jcb.151.3.709
3. Huangfu D, Liu A, Rakeman AS, et al. Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature. 2003;426(6962):83-87. doi:10.1038/nature02061
4. Taschner M, Lorentzen E. The Intraflagellar Transport Machinery. Cold Spring Harb Perspect Biol. 2016;8(10):a028092. doi:10.1101/cshperspect.a028092

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