Example Protocol III: Iodination of Crosslinkers. Thermo Fisher Scientific Pierce Pre-Coated Iodination Tubes
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4.
Remove sample from the reaction vessel to terminate the iodide oxidation.
5.
If no carrier iodide has been used in step 2, to enhance safety add NaI or KI to a final concentration of 1mM to the reaction mixture.
Note: If remaining non-reacted
125
I and small amounts of the active iodinating species interferes with subsequent experimental steps, tyrosine or other phenolic molecules, such as p -hydroxyphenylpropionic acid or p -hydroxyphenyl- acetic acid may be added to the mixture as iodination scavengers. Alternatively, reducing agents, such as sodium thiosulfate or sodium bisulfite, may be added at 1µmol. Excess iodide can be removed by gel filtration. Use a desalting column, such as Dextran Desalting Column (Product No. 43230), to separate protein.
125 I and scavenged 125 I from the iodinated
Example Protocol III: Iodination of Crosslinkers
The iodination of crosslinkers is performed rapidly, which limits hydrolysis of the NHS-ester portion of the crosslinker.
Perform the following steps in a darkened room if working with a photoreactive phenyl azide crosslinker, such as SASD.
A.
Materials Required
Iodinatable crosslinker
Phosphate Buffered Saline (PBS): 0.1 M sodium phosphate, 0.15 M NaCl; pH 7.2
(e.g., BupH™ Phosphate Buffered
Saline Pack, Product No. 28372).
Dimethylsulfoxide (DMSO)
Pierce Pre-Coated Tube
B.
Procedure
1.
Add 90µL of PBS to a Pierce Pre-Coated Iodination Tube.
2.
Dissolve 5.5µmol of crosslinker in 50µL of dry DMSO.
3.
Make a 1:20 dilution of the stock solution by adding 19µL of PBS to 1.0µL of stock solution and mix well. Immediately remove 10µL and add it to the tube containing the PBS and mix well (this solution will contain 55nmol of crosslinker).
This crosslinker working solution is not stable, therefore, proceed immediately to step 4.
4.
Add 40µCi Na
125
I and 18.5nmol potassium iodide (KI) in 10µL of 0.1M sodium phosphate, pH 7.4.
Note: KI is optional in this reaction and is used to increase the efficiency of iodine incorporation; however, including cold KI will decrease the specific activity of the crosslinker.
5.
Allow reaction to proceed for 30 seconds. Stop the reaction by removing the solution from the tube.
Note: Most applications will benefit from adding an iodine scavenger to prevent potential tyrosine iodination on the protein to be reacted with the crosslinker caused by excess reactive iodine. Scavengers such as 4-hydroxyphenylacetic
(or propionic) acid may be added (20nmol) for this purpose; do not use tyrosine nor reducing agents as they will interfere with the NHS-ester reaction.
6.
Immediately add the iodinated crosslinker into a tube containing 16nmol of protein in 300µL of borate buffer and allow to react for 30 minutes. Excess scavenger and iodide can be removed from the iodinated protein by gel filtration.
Depending on the downstream application, a separation step may not be required.
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