Important Product Information. Thermo Fisher Scientific Pierce Pre-Coated Iodination Tubes
Advertisement
Advertisement
Important Product Information
Exercise caution when working with radioactive iodide. Only appropriately trained personnel are to perform radioiodination procedures.
The efficiency of iodination is independent of pH values between 6 and 8.5. Efficiency decreases by ~15% at pH 5.0.
6
Pierce Iodination Reagent is virtually insoluble in aqueous media at pH 8.5. Extremely alkaline solutions will increase the solubility of the reagent.
Removal of the iodide solution from the Pre-Coated Test Tube terminates the oxidative production of the active iodinating species. After removal, the active iodinating species decays to molecular iodine within 15 minutes at room temperature.
6,7 After iodine incorporation, it is common to spike the iodinated sample with a carrier protein before gel filtration to remove excess radioactive iodine. To prevent iodination of the carrier protein, quench the solution containing oxidized iodide for active iodous species by adding a reducing agent or iodination scavenger, such as tyrosine, p -hydroxyphenyl propionic acid or p -hydroxyphenyl acetic acid, or allow it to decay to molecular iodine.
When proteins or peptides contain few available tyrosines for iodination, Sulfo-SHPP (Product No. 27712) is a useful reagent for introducing iodinatable groups onto lysine side chains or onto other primary amine-containing molecules.
Iodous ions (I + ) are produced by oxidation of iodide (I – the ortho ring position of tyrosine at neutral pH values.
) by Pierce Iodination Reagent and undergo electrophilic attack at
4,8
At pH 9, the imidazole ring of histidine can be labeled.
also been radiolabeled via the use of Pierce Iodination Reagent.
9
4
Mono- and di-iodinated derivatives of the tyrosine phenolic ring can be produced. The cytosine of DNA and RNA has
Example Protocol I: The Chizzonite Indirect Method for Iodination
Dr. Chizzonite used Pierce Pre-Coated Iodination Tubes (formerly called “IODO-GEN”) for indirect labeling of proteins and peptides. The Chizzonite method is the preferred and standard method for radiolabeling proteins and peptides. This method allows the sample to never contact the iodination reagent, which avoids the potential for oxidative damage to labile material as well as the potential for protein loss by aggregation or hydrophobic interaction with the coating. Furthermore, preactivating the radioactive iodide allows flexibility in the choice of reaction vessel to use. The preactivation protocol facilitates scaling up or down as needed.
A.
Materials Required
Tris Iodination Buffer 25mM Tris•HCl, pH 7.5, 0.4M NaCl. This buffer is used for iodide oxidation and for radioiodination of protein. Store buffer at 4°C.
Prepare 0.3-1.0nmol of protein in 100µL of Tris Iodination Buffer in a 1.5mL screw cap tube. Protein Stock Solution
High Tris Buffer (Optional) 0.125M Tris•HCl, pH 6.8, 0.15M NaCl. This buffer can be used in place of the standard Tris
Buffer where higher buffering capacity is needed for more alkaline solutions of Na 125 I.
Tris/BSA Buffer 0.25% bovine serum albumin (2.5mg/mL), 25mM Tris•HCl, pH 7.5, 0.4M NaCl, 5mM EDTA,
0.05% sodium azide. This buffer is used for blocking nonspecific binding sites on the desalting column, as a diluent for the radiolabeled protein, and as the wash buffer during desalting.
Tris/NaCl/EDTA Buffer
Scavenging Buffer
Desalting Column
25mM Tris•HCl, pH 7.5, 0.4M NaCl, 5mM EDTA, 0.05% sodium azide. This buffer can be used as a substitute for the Tris/BSA buffer for protein dilution and as the wash buffer during desalting when the inclusion of BSA as a carrier protein is not desired.
10mg tyrosine/mL in Tris Iodination Buffer or PBS, pH 7.4. This solution is saturating and crystals do not completely dissolve; p -hydroxyphenylpropionic acid or p -hydroxyphenylacetic acid can be used as substitutes for tyrosine.
10mL bed volume (e.g., Polyacrylamide Desalting Columns, 6K MWCO, Product No. 43243)
Pierce Biotechnology
3747 N. Meridian Road
PO Box 117
Rockford, lL 61105 USA
2
(815) 968-0747
(815) 968-7316 fax
Thermofisher.com

Public link updated
The public link to your chat has been updated.
Advertisement