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#3130: Procedure for Iodination of rat IGFBP-4
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. Rat IGFBP-4: 1 x 15 µg aliquot of Novozymes GroPep rat IGFBP-4 (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. 0.05 M sodium phosphate buffer, pH 7.2
6. Sodium metabisulphite Supplied by Sigma Chemical Company, St. Louis, MO, U.S.A.
7. Sephadex G75 Supplied by GE Healthcare (formerly Amersham Biosciences / Pharmacia Biotech), Uppsala, Sweden
chromatography medium
8. Chromatography buffer: RIA buffer : 50 mM Tris, 50 mM NaCl, 0.1% EDTA, 1% BSA (Fraction V Sigma 7888) and 0.5% Tween-20, pH 8.0.
Equipment
1. Laboratory equipped for safe handling of radioisotopes containing a fume hood with powered exhaust and lead shielding.
2. 0.9 x 45 cm glass chromatography column
3. Fraction collector (40 x 1 ml fractions required)
4. Timer
PREPARATION OF REAGENTS
Rat IGFBP-4: 1 x 15 µg Novozymes GroPep Rat IGFBP-4 (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 90 µl of 10 mM HCl to the vial. Ensure that all the rat IGFBP-4 is dissolved before proceeding.
3. Dispense 30 µl aliquots into eppendorf tubes.
4. Cap and seal with parafilm and store at -80°C. The IGFBP-4 so stored should be stable for 3 - 6 months.
5. For longer-term storage, freeze-dry the 5 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.
6. Alternatively, use a fresh 15 µg vial of rat IGFBP-4 from Novozymes GroPep for each iodination.
Na125I: Supplied by GE Healthcare (formerly Amersham Biosciences / Pharmacia Biotech), 1 mCi / 10 µl, 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).
0.05 M sodium phosphate, pH 7.2
1 Weigh out 0.78 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 7.2 with 1M NaOH.
4. Adjust the volume to 100 mls.
5. Prepare 10 ml aliquots for convenient use in iodinations. Store at -20°C.
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 commencing the iodination reaction
2. Add 10 mls of freshly made 0.05 M phosphate buffer pH 7.2 and mix (resultant concentration is 10 mg/ml).
3. In a 10 ml tube labelled as “Chloramine - T Working Solution”, place 100 µl of the 10 mg/ml solution and dilute with 4.9 ml of freshly made 0.05 M phosphate buffer pH 7.2.
4. Mix thoroughly. The “Chloramine - T Working Solution” is now ready for immediate use (0.2 mg/ml solution). Discard all Chloramine - T solutions when the iodination procedure is completed.
0.8 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 freshly made 0.05 M phosphate buffer pH 7.2 and mix (10 mg/ml).
3. Place 800 µl of the 10 mg/ml solution in a 10 ml tube and add 9.2 ml of freshly made 0.05 M phosphate buffer pH 7.2. Label as “sodium metabisulphite solution” (0.8 mg/ml).
4. Mix thoroughly. The solution is now ready for use. Discard all metabisulphite solutions when the iodination procedure is completed.
Chromatography buffer: Use RIA buffer
(50 mM Tris, 50 mM sodium chloride, 0.1% EDTA, 1% BSA (Fraction V Sigma A-7888))
To prepare 0.5 litre of chromatography buffer:
1. In a 500 ml beaker, add 3.025 g Tris base, 1.46 g NaCl and 0.5 g EDTA.
2. Add approximately 400 mls of distilled or Milli-Q water. Mix until dissolved.
3. Gradually add 5 g BSA (Fraction V Sigma A-7888) and allow to dissolve.
4. Adjust pH to 8.0 with 1M NaOH.
5. Pour solution into a 500 ml volumetric flask, and adjust volume to 500 mls with distilled or Milli-Q water. The buffer is now ready for use.
Sephadex G75 chromatography medium
1. Weigh out 3 g Sephadex G75 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 G75 chromatography column
All materials should be equilibrated at room temperature before use.
1. Set up a clean 0.9 x 45 cm glass 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 G75 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 rat IGFBP-4 to a specific activity of between 75 - 90 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 50mM phosphate buffer pH 7.2 to a 5 µg lyophilized aliquot of rat IGFBP-4 and let stand for 30 minutes at room temperature. Mix gently. The tube containing the reconstituted rat IGFBP-4 is now referred to as the “iodination reaction tube”.
2. Add approximately 0.7 mCi Na125 I (approximately 10 µl if the preparation is fresh) to the iodination reaction tube.
3. Add 20 µl of 0.2 mg/ml Chloramine-T Working Solution, and start the timer.
4. Gently mix the contents of the iodination reaction tube.
5. Stand for 6.5 minutes with periodic gentle mixing.
6. Stop the iodination reaction after 6.5 minutes by adding 10 µl 0.8 mg/ml sodium metabisulphite solution to the iodination tube. Mix gently. Then add 0.5 ml of the chromatography buffer.
7. Stand the tube for 5 minutes.
Isolation of [125I]-labelled rat IGFBP-4 from unreacted Na125I and other reaction products.
1. Load the contents of the iodination reaction tube onto the equilibrated column.
2. Run the column at a flow rate of 0.25 ml/minute and collect forty x 1 ml fractions.
Selection of the fraction containing the desired [125I ] - labeled rat IGFBP-4.
1. Plot the gamma radioactivity of each fraction versus the fraction number.
1. Count 10 µl of each fraction in a gamma counter and plot against fraction number.
2. Plot the data on a graph showing the gamma radioactivity per 10 µl of each fraction number.
3. The labeled rat IGFBP-4 elutes in the 18-19 ml fractions and unincorporated radioactivity in the 35-37 ml fractions.
4. Calculate the percent incorporation of [125I] and determine the specific activity of the rat IGFBP-4.
5. Dilute the peak tubes in chromatography (RIA) buffer to give ~3 x 106 cpm / ml.
6. Aliquot into convenient sized aliquots, commensurate with the planned use of tracer.
7. Store at -20°C. Avoid repeated freeze-thawing of aliquots. The rat IGFBP-4 tracer is stable for approximately 3 months.
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