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INSTRUCTIONS
Pierce Pre-Coated Iodination Tubes
28601
Number
28601
MAN0016379
Rev. A.0
Pub. Part No. 2160692.5
Description
Pierce Pre-Coated Iodination Tubes, 10 borosilicate test tubes (12 75mm) coated with 50µg Pierce
Iodination Reagent (formerly called “IODO-GEN” Iodination Reagent)
Tube-to-tube variation: 5%
Storage: Upon receipt store at room temperature protected from moisture. After opening barrier pouch, close the interior resealable bag containing the tubes with the yellow closures and desiccant.
Table of Contents
Introduction ................................................................................................................................................................................. 1
Important Product Information .................................................................................................................................................... 2
Example Protocol I: The Chizzonite Indirect Method for Iodination .......................................................................................... 2
Example Protocol II: Direct Method for Iodination ..................................................................................................................... 3
Example Protocol III: Iodination of Crosslinkers ........................................................................................................................ 4
Cited References .......................................................................................................................................................................... 5
General References ...................................................................................................................................................................... 5
Introduction
The Thermo Scientific™ Pierce™ Pre-Coated Iodination Tubes are a ready-to-use source of Pierce Iodination Reagent that provides convenient, consistent radioiodination of proteins and cell membranes.
1 Pierce Iodination Reagent (1,3,4,6tetrachloro-3 , 6 -diphenylglycouril; formerly called “IODO-GEN” Iodination Reagent) is an effective and mild oxidation reagent. This reagent is as effective as enzymatic methods for iodination of externally exposed residues and as effective as chloramine-T for general protein iodination, in addition to being more mild retained cell viability.
2
1-5 resulting in limited oxidative damage and
During oxidative reactions in the Pre-Coated Tubes, which are coated at the bottom with about 100µL, the iodination reagent remains adherent to the vessel because it is insoluble in typical aqueous media. This feature enables the sample to be decanted from the coated vessel to terminate the oxidative reaction. Although Pierce Iodination Reagent can be coated to any borosilicate glass surface, coating and drying procedure is very difficult to achieve in the typical laboratory without the reagent becoming inactive or flaking from the surface. The ability of the reagent to adhere tightly to a vessel depends on temperature, humidity, solvent and evaporation rate, and the coating procedure can be time-consuming, troublesome and difficult to reproduce. We have optimized these conditions in preparing the Pre-Coated Iodination Tubes, providing a readyto-use product for convenient iodination of typical protein and peptide samples.
Pierce Pre-Coated Iodination Tubes eliminate the troublesome coating step and offers a tightly adherent film of iodination reagent that will not flake off the tube walls. Each tube is consistently coated and exhibits less than 5% variation from tubeto-tube. The consistent coating results in highly reproducible radioiodinations.
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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)
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PO Box 117
Rockford, lL 61105 USA
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B.
Procedure
1.
Wet a Pierce Pre-Coated Iodination Tube with 1mL of Tris Iodination Buffer and decant.
2.
Add 100µL of Tris Buffer to the pre-coated tube. Add buffer directly to the bottom of the tube; do not allow buffer to drain down the tube wall.
3.
Add 10µL (1.0mCi) of Na
125
I (e.g., from MP Biomedicals or other supplier) and mix.
Note: The amount of Na 125 I may be increased to 20µL for labeling larger amounts of protein or to increase the radiospecific activity of the iodinated protein.
4.
Allow iodide to activate for 6 minutes at room temperature. Swirl the tube every 30 seconds.
5.
Remove and add the activated iodide to the protein solution and mix.
6.
React for 6-9 minutes at room temperature. Mix by gently flicking the tube every 30 seconds.
7.
Add 50µL of Scavenging Buffer. Mix and incubate for 5 minutes with additional flicking at 1 and 4 minutes.
8.
Add 1.0mL of Tris/BSA Buffer and mix. Remove a 5µL aliquot for determination of total 125 I incorporation.
(If BSA is omitted from the reaction, use 1.0mL of Tris/NaCl/EDTA Buffer.)
9.
Add sample to a 10mL bed volume desalting column that has been equilibrated with 20mL of Tris/BSA Buffer.
Note: If the protocol is being modified to omit BSA as a carrier protein, after column equilibration with Tris/BSA
Buffer, add 20mL of Tris/NaCl/EDTA Buffer.
10.
Wash tube containing the iodinated protein with 0.5mL of Tris/BSA Buffer and add this to the column to increase recovery of the iodinated protein. Use 0.5mL of Tris/NaCl/EDTA Buffer if BSA is omitted.
11.
Recover sample by adding 1.0mL aliquots of Tris/BSA Buffer. Collect 6 × 1mL fractions in 12 × 75mm polypropylene tubes. If BSA is omitted, use 0.5mL of Tris/NaCl/EDTA Buffer. Scan eluted fractions for radioactivity.
12.
After the last fraction has emerged from the column, seal the tip, recap column and dispose column as radioactive waste.
13.
Determine protein bound and free 125 I by standard TCA precipitation procedures.
14.
Determine radiospecific activity of the labeled protein. Typically, the radiospecific activity will be 2500-4000cpm/fmole when 0.3nmole of protein is labeled by this procedure. The radiospecific activity can be manipulated to vary from 500-
7500cpm/fmol by using higher or lower amounts of protein or 125 I-Na in the protocol.
15.
Dilute the labeled protein with Tris/BSA buffer to an activity of 1-2 ×10 8 cpm/mL and store at 4°C. Labeled antibodies are usually stable for up to eight weeks; very labile proteins may retain stability for 3-4 weeks.
Example Protocol II: Direct Method for Iodination
The following protocol is based on the method by Mary Ann K. Markwell.
2 Additional notes and suggestions are provided to address commonly asked questions and variations. In this method, the material to be iodinated is added directly to the Pierce
Pre-Coated Iodination Tube (formerly called “IODO-GEN” Pre-Coated Iodination Tube).
1.
Dissolve the sample in an appropriate buffer and add to a pre-coated tube.
Note: Choose a buffer and temperature that is compatible with the specific sample and biological system. The optimal temperature and pH range is 0-37°C and 6-8.5, respectively. For cells a medium, such as Dulbecco’s PBS plus glucose, may be used to maintain cells during iodination. Avoid using media containing tyrosine or serum, as most sera contain appreciable amounts of NaI. Also avoid 2-mercaptoethanol, dithiothreitol and cysteine.
2.
Add carrier-free Na 125 I to the solution in the pre-coated tube. Typically, 500µCi of Na 125 I is used per 100µg of protein.
Note: Carrier NaI or KI is sometimes used to drive the reaction, to label inner membrane proteins, or for safety reasons.
However, some of the iodine incorporated will not be radioactive. Carrier iodide from 0.25mM to 1mM may be used with 0.5-1.0mCi of radioiodide. If a “hot” label is required, use more carrier-free radioiodide or omit the non-radioactive carrier iodide.
3.
Allow the reaction to proceed for 10-15 minutes. If the sample is stable and a higher specific radioactivity is desired, the reaction time may be increased. Agitate the vessel for best results. Perform a time study to optimize the procedure.
Shorter reaction times may be necessary if the sample is labile.
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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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PO Box 117
Rockford, lL 61105 USA
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Cited References
1.
Fraker, P.J. and Speck, J.C., Jr. (1978). Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a, 6adiphenylglycouril. Biochem Biophys Res Commun 80(4): 849-57.
2.
Markwell, M.A.K. and Fox, C.F. (1978). Surface-specific iodination of membrane proteins of viruses and eucaryotic cells using 1,3,4,6-tetrachloro-3a,
6a-diphenylglycouril. Biochem 17: 4807-17.
3.
Salacinski, P., et al.
(1979). A new, simple method which allows theoretical incorporation of radioiodine into proteins and peptides wihout damage . J
Endocrinol 81(2): 131P.
4.
Salacinski, P.R.P., et al.
(1981). Iodination of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, 1,3,4,6-tetrachloro-3 , 6 dipenyl glycoluril (IODO-GEN ® ). Anal Biochem 117: 136-46.
5.
Goding, J.W. (1986). Monoclonal Antibodies: Principles and Practice . Academic Press, London, 142-56.
6.
Paus, E., et al.
(1982). Radioiodination of proteins with the IODO-GEN method. in Radioimmunoassay in Related Procedures in Medicine; Proc Int
Symp 161-71.
7.
Bailey, G.S. (1996). The Iodogen method for radiolabeling protein. in The Protein Protocols Handbook , J. M. Walker, ed., Humana Press, Inc.,
Totowa, New Jersey. 673-4.
8.
Seevers, R.H., and Counsell, R.E. (1982). Radioiodination techniques for small organic molecules. Chem Rev 82: 575-90.
9.
Piatyszek, M.A. et al.
(1988). IODO-GEN-mediated radioiodination of nucleic acids. Anal Biochem 172: 356-9.
General References
Alexander, N. M. (1973). Oxidation and oxidative cleavage of tryptophanyl peptide bonds during iodination. Biochem Biophys Res Commun 54(2): 614-21.
Alexander, N. M. (1974). Oxidative cleavage of tryptophanyl peptide bonds during chemical- and peroxidase-catalyzed iodinations.
J Biol Chem 249: 1946-
52.
Chen, T., et al.
(1996). Interaction of phosphorylated FceRIg ITAM-based peptides with dual and single SH2 domains of p72Syk: Assessment of binding parameters and real time binding kinetics. J Biol Chem 271: 25308-15.
Chizzonite, R., et al.
(1991). IL-12: Monoclonal antibodies specific for the 40kDa subunit block receptor binding and biologic activity on activated human lymphoblasts. J Immunol 147: 1548-56.
Chizzonite, R., et al.
(1992). IL-12 Receptor: I. Characterization of the receptor on PHA-activated human lymphoblasts. J Immunol 148: 3117-24.
Chua, A.O., et al.
(1994). Expression cloning of a human IL-12 receptor component: A new member of the cytokine receptor superfamily with strong homology to gp130. J Immunol 153: 1320-7.
Schechter, Y., et al . (1975). Selective oxidation of methionine residues in proteins.
Biochemistry 14: 4497-503.
Desai, B. B., et al.
(1992). IL-12 Receptor: II. Distribution and regulation of receptor expression. J Immunol 148: 3125-32.
Gately, M.K. and Chizzonite, R., (1992). Measurement of human and mouse interleukin-12. in Current Protocols in Immunology, (J.E. Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, Eds.), John Wiley and Sons, New York, N.Y.
Greenfeder, S., et al.
(1995). Expression cloning of a cDNA encoding a novel murine interleukin-1 receptor accessory protein. J Biol Chem 270: 13757-65.
Mallamaci, M.A., et al. (1993). Identification of sites on the human FceRI alpha-subunit which are involved in binding human and rat IgE. J Biol Chem
268: 22076-83.
Riske, F., et al.
(1991). High affinity Human IgE Receptor (FceRI): Analysis of the functional domains of the alpha-subunit with monoclonal antibodies. J
Biol Chem 266: 11245-51.
Van der Laken, C.J. et al.
(1997). Preferential localization of systemically administered radiolabeled interleukin 1 in experimental inflammation in mice by binding to the type II receptor. J Clin Invest 100(12): 2970.
Wood, W. G. and Scriba, P.C. (1981). Experiences using chloramine-T and 1, 3, 4, 6, tetrachloro-3 , 6 -diphenylglycoluril (IODO-GEN ® radioiodination of materials for radioimmunoassay. J Clin Chem Clin Biochem 19: 1051-6.
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Pierce Biotechnology
3747 N. Meridian Road
PO Box 117
Rockford, lL 61105 USA
5
(815) 968-0747
(815) 968-7316 fax
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