Preparation of FSL-Tyr1 Construct Protocol

FSL-Tyr1

Catalog Number: F7558
Storage: Temperature –20 °C
Synonym: FSL-Tyr1(forY)-SC2-L1

Product Description

Molecular formula: C₉1H₁₄₀N₁₆O₃₃PS
Molecular weight: 2017.15

FSL-Tyr1 is a KODE™ technology construct designed to label hydrophobic surfaces including living cells and virions with ¹²⁵I. All KODE FSL constructs consist of three essential designable features:

• functional component (F)
• spacer (S)
• diacyl lipid (L)

FSL-Tyr1 is comprised predominantly of an N-formyltyrosine monomer, which can be radiolabeled with ¹²⁵I, representing F, conjugated via a carboxymethylglycine based linker (SC2) to an activated adipate derivative of dioleoylphosphatidylethanolamine (L). All FSL constructs disperse in biocompatible media, and spontaneously and stably incorporate into cell/virion membranes. Cells and virions modified with KODE constructs are known as kodecytes¹ or kodevirions,² respectively, and usually maintain their normal vitality and functionality. Following ¹²⁵I radiolabeling, FSL-Tyr1 has been specifically designed to create ¹²⁵I labeled kodecytes or kodevirions.²

Reagents Required but Not Provided for ¹²⁵I Radiolabeling Procedure

• Iodination tubes (Cat. 28601, Thermo Fisher Scientific Inc.)
• Na¹²⁵I (Cat. NEZ-033A, Perkin-Elmer)
• Amicon Ultra MWCO10kDa filter device (Cat. Z677108)
• Tris-HCl-NaCl buffer (25 mM Tris-HCl, pH 7.5, with 0.4 M NaCl)
• Phosphate buffered saline (Cat. P5493)

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Preparation Instructions

Stock Solution is prepared by reconstituting the product at a concentration of 2 mg/mL in saline or PBS. Buffered solutions are preferred for long-term storage. The product should not be reconstituted in water, unless used immediately as the product is unstable when stored in water. The 2 mg/ml Stock Solution can be frozen in aliquots for later usage. Thawed product should be briefly sonicated before use.

Storage/Stability

Store unopened product at –20 °C. Store the Stock Solution in aliquots at –20 °C. Avoid repeated freezing and thawing of solutions. Solutions in PBS, pH 7, can be stored at 2–8 °C for up to 2 weeks.

Procedure - Iodination of FSL-tyrosine to FSL-¹²⁵I

1. Wet interior surface of iodination tube with 1 mL of Tris-HCl-NaCl buffer and decant.
   
   
2. Add 100 µL of Tris-HCl-NaCl buffer to the tube, followed by 1 mCi of Na¹²⁵I. Incubate at room temperature for 6 minutes with swirling every 30 seconds.
   
   
3. Transfer the activated Na¹²⁵I solution to a new centrifuge tube containing 150 µL of FSL-Tyr1 Stock Solution (0.3 mg). Incubate at room temperature for 8 minutes, with swirling every 30 seconds.
   
   
4. Transfer reaction solution to a Amicon Ultra-10 filter device. Rinse the reaction tube with 250 µL of Tris-HCl-NaCl buffer and add to reaction solution. Centrifuge the filter device for 30 minutes at 15,000 x g. Collect flow-through solution.
   
   
5. Rinse the reaction tube with an additional 300 µL of Tris-HCl-NaCl buffer and add to Amicon Ultra-10 filter device. Centrifuge for 30 minutes at 15,000 x g. Collect second flow-through solution. The flow-through solutions contain the radiolabeled FSL-Tyr1 (FSL-¹²⁵I).
   
   
6. Quantify all fractions using a dose calibrator prior to pooling, then aliquot solution and store at –20 °C. FSL radioiodination can be confirmed by SDS-PAGE on a 15% acrylamide gel in non-reducing sample buffer.

Cell/Virion labeling

1. Add 1 volume of FSL-¹²⁵I (1–100 µg/mL diluted in PBS) to 1 volume of cells/virions. Incubate for 1 hour at 37 °C.
   
   
2. Wash with PBS or other appropriate buffer (may be optional). Store kodecytes/kodevirions in serum free medium.
   
   Note: Rate of FSL insertion is primarily determined by FSL concentration, incubation time, and temperature.