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#3001: Procedure for the Iodination of IGFs
Note that the operator should be trained in the safe use and handling of radioisotopes and hold an appropriate licence if required by the local radiation authority.
MATERIALS
Reagents
1. Novozymes GroPep IGF Peptide: 1 x 100 µg Receptor Grade IGF-I (chicken, bovine, human, porcine, or rat) or IGF-II (chicken, bovine, human, porcine, or rat) (lyophilized)
2. Na125I: Supplied by GE Healthcare (formerly Amersham Biosciences / Pharmacia Biotech), 1 mCi/10µl, 10 - 20 mCi/µg
3. Chloramine-T: Supplied by Sigma Chemical Company, St. Louis, MO, U.S.A.
4. 10 mM HCl
5. 0.5 M sodium phosphate, pH 7.5
6. sodium metabisulphite Supplied by Sigma Chemical Company, St. Louis, MO, U.S.A.
7. Chromatography buffer: 50 mM sodium phosphate containing 150 mM sodium chloride and 0.25% (w/v) Bovine Serum Albumin (RIA Grade), pH 6.5
8. Sephadex G50 fine chromatography medium Supplied by GE Healthcare (formerly Amersham Biosciences / Pharmacia Biotech), Uppsala,
Sweden
9. 99% (w/v) trichloracetic acid (TCA)
Equipment
1. Laboratory equipped for safe handling of radioisotopes containing a fume hood with powered exhaust and lead shielding.
2. 1 x 25 cm glass or plastic chromatography column
3. Fraction collector (60 x 2 minute fractions required)
4. Pump (0.25 ml/min required)
5. 60 second timer
PREPARATION OF REAGENTS
Novozymes GroPep IGF Peptide: 1 x 100 µg Receptor Grade IGF-I (chicken, bovine, human, porcine, or rat) or IGF-II (chicken, bovine, human, porcine, or rat) (lyophilized)
1. Remove the metal cap from the vial and carefully loosen the bung to equalize the slight vacuum within the vial to prevent any loss of peptide upon opening.
2. Add 100 µl of 10 mM HCl acid to the vial. Ensure that all the peptide is dissolved before proceeding.
3. Dispense 10 µl aliquots into eppendorf tubes.
4. Cap and seal with parafilm and store at -80°C. IGFs Hormone so stored should be stable for 3 - 6 months.
5. For longer-term storage, freeze-dry the 10 µg aliquots and store at -80°C. Lyophilised aliquots will be stable for at least 3 years if stored at -80°C. Use for iodination when required.
7. Alternatively, use a fresh 20 µg vial of IGF from Novozymes GroPep for each iodination.
For further handling information consult:
#1000: Handling of Novozymes GroPep IGF-I, IGF-II and IGF Analogs
Na125I: Supplied by GE Healthcare (formerly Amersham Biosciences / Pharmacia Biotech), 1 mCi/10µl, 10 - 20 mCi/µg
1. Order Na125I from GE Healthcare (formerly Amersham Biosciences / Pharmacia Biotech), Catalog number IMS30, approximately 100 mCi/ml, 10 - 20 mCi/µg
2. Record batch / lot number, pH, reference date, activity and specific activity.
3. On the day of iodination, calculate the volume of Na125I to add to the iodination reaction given that 1 mCi of radioactive label should be added. The reference date and the specific activity of the Na125I will affect the volume of solution that is required. (Note that the half-life of 125I is 60 days. Refer to GE Healthcare (formerly Amersham Biosciences / Pharmacia Biotech), "Table of Decay of 125I" included in the Na125I pack).
Oxidant: Chloramine-T (available from Sigma Chemical Co., St. Louis MO, USA, Cat # C9887, (250 g))
1. Weigh out 0.1 g Chloramine-T and place in a 10 ml tube.
The following steps should only be completed immediately before commencement of the iodination reaction.
2. Add 10 mls distilled or Milli-Q water and mix (resultant concentration is 10 mg/ml).
3. In a 10 ml tube labelled as “Chloramine - T Working Solution”, place 400 µl of the 10 mg/ml solution and dilute with 9.6 ml of distilled or Milli-Q water.
4. Mix thoroughly. The “Chloramine - T Working Solution” is now ready for immediate use (0.4 mg/ml solution). Discard all Chloramine - T solutions when the iodination procedure is completed.
10 mM HCl
0.5 M sodium phosphate, pH 7.5
1. Weigh out 7.8 g sodium monophosphate dihydrate (NaH2PO4.2H2O) and place in a 100 ml beaker.
2. Add 90 mls distilled or Milli-Q water to the beaker and mix to dissolve.
3. Adjust the pH to 7.5 with 1M NaOH.
4. Adjust the volume to 100 mls.
5. Prepare 0.5 ml aliquots for convenient use in iodinations. Store at -20°C.
0.6 mg/ml sodium metabisulphite (Na2S2O5)
1. Weigh out 0.1 g of sodium metabisulphite and place in a 10 ml tube.
The following steps should only be completed immediately before commencing the iodination reaction.
2. Add 10 ml distilled or Milli-Q water and mix (10 mg/ml).
3. Place 600 µl of the 10 mg/ml solution in a 10 ml tube and add 9.4 ml of distilled or Milli-Q water. Label as “sodium metabisulphite solution” (0.6 mg/ml).
4. Mix thoroughly. The solution is now ready for use. Discard all metabisulphite solutions when the iodination procedure is completed.
Chromatography buffer:
(50 mM sodium phosphate buffer containing 150 mM sodium chloride and 0.25% (w/v) BSA, pH 6.5)
To prepare 0.5 litre of chromatography buffer:
1. In a 500 ml beaker, add 3.9 g sodium dihydrogen orthophosphate dihydrate (NaH2PO4.2H2O), 4.383g NaCl and 1.25 g BSA (Sigma Chemical Co., RIA Grade).
2. Adjust pH to 6.5 with 1M NaOH.
3. Pour solution into a 500 ml volumetric flask, and adjust volume to 500 mls with distilled or Milli-Q water.
4. Prepare a 20% (w/v) sodium azide (NaN3) solution. In a fume hood add 20 g NaN3 to 100 mls of distilled or Milli-Q water.
5. Add 0.5 ml of the 20% (w/v) NaN3 solution to the prepared chromatography buffer. The final concentration of NaN3 in the buffer is therefore approximately 2% (w/v). The buffer is now ready for use.
Sephadex G50 fine chromatography medium
1. Weigh out 3 g Sephadex G50 chromatography gel (GE Healthcare (formerly Amersham Biosciences / Pharmacia Biotech), Uppsala, Sweden).
2. To pre-swell the gel, add approximately 200 mls of distilled or Milli-Q water. Excess water is added to allow the gel to swell without the possibility of drying out.
3. Allow to stand for 3 hours at 80°C or overnight at room temperature.
4. Once pre-swelling has occurred, allow to settle and then pour off the gel slurry supernatant to remove the gel fines. (Failure to remove the gel fines may cause the chromatography column to become blocked during use).
5. To ensure that the gel fines are removed, resuspend the gel in 200 mls distilled or Milli-Q water, allow to settle, and pour off the gel slurry supernatant as before. Repeat until no more fines are evident.
6. Resuspend in 20 mls distilled or Milli-Q water. The gel is now ready for pouring into a column.
PROCEDURES
Preparation and equilibration of the G50 chromatography column
All materials should be equilibrated at room temperature before use.
1. Set up a clean 1 x 25 cm glass or plastic chromatography column, ensuring that the column is vertical, and well clamped into position. Add enough distilled or Milli-Q water to occupy about 20% of the column volume and drain to about 10% of column volume to remove air from the column fittings.
2. Mix the pre-swollen Sephadex G50 gel so that the gel slurry is homogeneous. Carefully pour the gel slurry into the column. Note that the gel slurry should not be too thick for pouring as air bubbles will become trapped in the column.
3. Equilibrate the column with the chromatography buffer (prepared as indicated above). Ensure that the buffer is at room temperature before use to avoid air bubbles forming in the column. It is suggested that a minimum of three column volumes (approximately 60 mls) are run through the column to block all non-specific binding sites on the column.
Iodination Reaction
Please note that this iodination procedure is designed to label Novozymes GroPep IGF peptides to a specific activity of between 50 - 80 Ci/g. Note also that the iodination reaction must be completed in a Type B laboratory or certified iodination laboratory in a fume hood.
1. Add 50 µl of 10mM HCl to a 10 µg lyophilized aliquot of IGF peptide and let stand for 30 minutes at room temperature. Mix gently. The tube containing the reconstituted peptide is now referred to as the “iodination reaction tube”.
2. Add 50 µl 0.5 M sodium phosphate pH 7.5 to the iodination reaction tube.
3. Add approximately 1 mCi Na125 I (approximately 10 µl if the preparation is fresh) to the iodination reaction tube.
4. Add 20 µl of 0.4 mg/ml Chloramine-T Working Solution, and start the timer.
5. Gently mix the contents of the iodination reaction tube.
6. Stand for 60 seconds only.
7. Add 20 µl 0.6 mg/ml sodium metabisulphite to the iodination tube; mix gently.
8. Stand the tube for 5 minutes.
Isolation of [125I] - labelled IGF from unreacted Na125I and other reaction products.
1. Dilute the iodination reaction mixture with 300 µl chromatography buffer.
2. Load onto the equilibrated column; wash into the column with 3 x 500 µl of chromatography buffer to allow the sample to enter the column bed. This washing-in step is important!
3. Run the column at a flow rate of 0.25 ml/minute and collect 0.5 ml fractions for 2 hours. i.e., 60 x 0.5 ml fractions.
Selection of the fraction containing the desired [125I] - labelled IGF peptide.
The fractions which contain the [1251] - labelled IGF peptide and whose radioactivity is greater than 95% TCA precipitable are chosen for use.
A. Plot the gamma radioactivity per 5 µl of each fraction versus the fraction number.
1. Number another set of tubes equal to the number of fractions collected. Add a 5 µl sample from each fraction to the corresponding numbered tube. Dilute with 900 µl of chromatography buffer.
2. Count each diluted sample tube for gamma radioactivity.
3. Plot the data on a graph showing the gamma radioactivity per 5 µl of each fraction number.
B. Determine the percentage of TCA precipitable radioactivity contained in each fraction.
1. After counting the diluted sample tubes, add 100 µl of 99% (w/v) TCA to each tube. Vortex and let stand on ice for a minimum of 30 minutes to maximize protein precipitation.
2. Centrifuge for 10 minutes at 4,000 g.
3. Remove the supernatant from each test tube, and transfer to another series of labelled test tubes.
Care should be taken as the protein pellet is relatively fragile.
4. Count the supernatant fraction for gamma radioactivity.
5. The percentage of each fraction that is TCA precipitable is the ratio between the radioactivity contained in the pellet and the sum of the radioactivity in the pellet plus the supernatant. This is termed the percent TCA - precipitable.
6. Plot the TCA - precipitable radioactivity percentage for each fraction on the same graph as the [125I] gamma radioactivity.
7. The fractions whose radioactivity is greater than 95% TCA precipitable will contain the [125I] - labelled IGF peptide. (Do not include the aggregate fraction which may be the first small peak of radioactivity seen).
8. Pool the chosen fractions and aliquot into convenient sized aliquots, commensurate with the planned use of tracer.
9. Store at -20°C. Avoid repeated freeze-thawing of aliquots.
© Novozymes GroPep Limited
28 Dalgleish Street
Thebarton SA 5031
PO Box 10065 BC
Adelaide SA AUSTRALIA
Telephone +61 8 8354 7700
Facsimile +61 8 8354 7788
Email:
AUAD-bioreagents@novozymes.com
Website: www.gropep.com.au
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