Radiolabelling of soluble proteins

4.9 Radiolabelling of soluble proteins

The methods in this section vary in the harshness (potential for alteration of the conformation of the labelled material) of the reaction required to achieve the desired result.

Chloramine T and Iodogen labelling involve tyrosyl residues predominantly; if they inactiv- ate antibody binding then try labelling onto lysyl residues. It is essential that use of radiolabels is

C H A P T E R 4: Antibodies as probes C H A P T E R 4: Antibodies as probes

4.9.1 Chloramine T method

In alkaline conditions, chloramine T is slowly converted to hypochlorous acid which acts as an oxidizing agent. At pH < 8.0, oxidation results in iodine incorporation into tyrosine residues, but at a higher pH histidine also becomes labelled. The method is the best one to try first for labelling antigens or antibodies, either in solution or bound to a solid-phase immunoadsorbent to protect the active site.

MATERIALS AND EQUIPMENT All reagents should be prepared just before labelling.

0.1 M tris(hydroxymethyl)-aminomethane (Tris)–HCl buffer, pH 7.4 Protein for iodination (500 µg/ml in Tris–HCl buffer) Chloramine T (1 mg/ml in Tris–HCl buffer) Sodium metabisulphite (2 mg/ml in Tris–HCl buffer) Potassium iodide (5 × 10 − 5M in Tris–HCl buffer) Sodium 125

I, carrier free Phosphate-buffered saline (PBS), containing 0.25% w/v gelatin Sephadex G-25 Disposable chromatography column (e.g. a disposable pipette plugged with glass wool) Gamma spectrometer Caution: Wear gloves when handling glass wool.

METHOD

I and 10 µl of chloramine T (1 mg/ml initial concentration).

1 Mix 100 µl of protein (500 µg/ml initial concentration) with 18.5 × 10 6 Bq 125

2 Incubate for 2–4 min at room temperature.

3 Add 10 µl of sodium metabisulphite solution (2 mg/ml initial concentration) and mix thoroughly.

4 After 2 min, add 10 µl of potassium iodide solution.

5 Separate the labelled protein from the free iodine using a column of Sephadex G-25 equilibrated with PBS containing 0.25% gelatin.

6 Elute the column with PBS containing gelatin and collect 0.5-ml fractions.

7 Determine the c.p.m. of each fraction using a g spectrometer. Identify the first peak of radioactivity athis contains the labelled protein.

8 Store at 4°C for use.

TECHNICAL NOTES • Proteins denature readily at low concentration, therefore gelatin or bovine serum albumin (BSA)

should be incorporated in the elution buffer. The gelatin or BSA is also necessary to prevent non- specific binding of protein to the Sephadex. If labelled proteins without carrier protein are needed, the column can be precycled with gelatin or BSA to block non-specific uptake and the column used with buffer alone.

4.9RADIOLABELLING OF SOLUBLE PROTEINS

• Although the Sephadex G-25 column is adequate to rapidly separate the protein from the harmful reaction reagents and the free iodine, the labelled protein often needs further purification. Frequently, labelled proteins show some non-specific ‘stickiness’ due to protein aggregates. These can be removed by gel filtration on Sephadex with an appropriate fractiona- tion range. The nascent formation of unrelated immune complexes at equivalence within the labelled protein solution is also an effective way of removing non-specific binding, but must be used with care in case the complex components interfere with the subsequent assay.

• This labelling technique may be used to iodinate antibodies attached to an antigen immunoad- sorbent (this protects the antigen-binding site). The free iodide is removed by washing with buffer, and the labelled antibody is recovered by acid elution (see Section 1.4.2).

4.9.2 Determination of specific activity of radiolabelled protein

The protein concentration of the labelled material changes during radiolabelling and one cannot assume that all of the radioactive iodine is covalently bound to the protein.

Total radioactivity (in c.p.m.) is a less useful measure of the efficiency of labelling than specific activity (c.p.m./mg protein). It is necessary therefore to determine the new protein concentra- tion, either by its UV absorbance if it is a pure protein (see Appendix B for extinction coefficients of common immunochemicals), or by the Bradford method if it is a mixture of proteins (see Appendix B.5.1 and B.5.2).

MATERIALS AND EQUIPMENT Radioiodinated sample

Glass-fibre filter discs, e.g. Whatman GF/A Large mapping pins in a cork board Automatic pipette, 1–5 µl 10-ml glass test tubes Ice-cold 10% w/v trichloroacetic acid (TCA) Absolute ethanol Gamma spectrometer

METHOD

1 Mount each filter on a map pin (or hypodermic syringe needle) so that it is held clear of the cork board.

2 Dispense between 1 and 5 µl of each sample (this should correspond to approximately 10–50 000 c.p.m. of the total radioactivity) onto two separate filter discs and transfer one into a 10-ml test tube.

3 Add 2 ml of ice-cold TCA to the tube, mix by vortexing and leave for 10 min.

4 Decant the TCA and replace with 2 ml of ethanol.

5 Mix by vortexing and leave for 10 min.

6 Transfer each pair of TCA-treated and -untreated filters to separate small plastic tubes and determine their radioactive content in a gamma spectrometer.

C H A P T E R 4: Antibodies as probes

Calculation of labelling efficiency

c.p.m. in TCA-treated sample

% Protein-bound radioactivity =

total c.p.m. in untreated sample

Specific activity (c.p.m./mg) = c.p.m. in TCA-treated sample × 1000

protein concentration ( g ml µ / ) TECHNICAL NOTES

volume of treated sample ( l) µ

• Proteins labelled to a very high specific activity (e.g. for autoradiography or immunoprecipita-

tion) can give problems in this procedure: often it is not possible to measure accurately a sufficiently small aliquot to obtain a c.p.m. within the spectrometer range and the protein con- centration can be too low for efficient TCA precipitation. Both these problems can be overcome by prior dilution of an aliquot of the labelled protein in bovine serum albumin (1 mg/ml) dissolved in phosphate-buffered saline.

• The same principles apply for the measurement of any radioactive label bound to a protein

molecule, with modifications to take account of the nature of the isotope. Clearly, if the

radioisotope is a low-energy emitter ( 3 H, 14 C or 35 S) then a b spectrometer should be sub- stituted for the g spectrometer. • Treating the filter with ethanol not only aids the removal of TCA-soluble material but also

speeds drying. Low-energy emitters need liquid scintillants, many of which will not tolerate water.

4.9.3 Iodination of proteins with Iodogen

The main damaging reaction in the chloramine T method is the exposure of proteins to the oxid- izing agent. The use of the insoluble chloroamide 1,3,4,6-tetrachloro-3a, 6a-diphenylglycoluril (Iodogen) coated onto the surface of the reaction tube, or a plastic bead, reduces denaturation of antigen or antibodies during iodination.

Preparation in advance

MATERIALS AND EQUIPMENT Iodogen

Dichloromethane Test tube, 10 × 75 mm solvent-resistant plastic (e.g. polypropylene) Water bath Nitrogen cylinder

METHOD

1 Prepare a solution of Iodogen (0.1 mg/ml) in dichloromethane.

2 Add 0.2 ml of this solution (20 µg Iodogen) to a test tube.

3 Evaporate the methylene chloride in a stream of nitrogen, while rotating the tube slowly in

a water bath at 37°C, to leave a thin film of Iodogen in the bottom of the tube.

4 Store in the dark at –20°C. The stored tubes may be used for several weeks.

4.9RADIOLABELLING OF SOLUBLE PROTEINS

Iodination technique

MATERIALS AND EQUIPMENT Protein for iodination (1 mg/ml) in borate–saline buffer, pH 8.3, ionic strength 0.1 Sodium 125

I, carrier free Iodogen-coated tubes, prepared as above

METHOD

1 Place the Iodogen-coated tube on ice and add 0.2 ml of protein solution (1 mg/ml initial concentration).

2 Initiate the reaction by the addition of 10 µl of sodium 125

I solution (37 × 10 6 Bq l25 I).

3 Incubate for 5 min with gentle stirring.

4 Terminate the reaction by decanting the protein solution and leave for 10 min to allow reactive iodine to decay.

5 Separate the labelled protein from the free iodine by gel filtration as for the chloramine T method.

6 Determine the protein-bound radioactivity and specific activity.

TECHNICAL NOTES • This method has been found to be more efficient than the chloramine T reaction for some

proteins but is inferior for others. The labelling technique of choice must be determined by experimentation.

• A similar method may be used for the iodination of cells. Typically, use 4 × 10 6 cells, 6.6 µg of

I in a total volume of 400 µl of phosphate-buffered saline (PBS). Add mixture to a tube coated with 50 µg Iodogen. Incubate for 15 min on ice with gentle stirring. Terminate the reaction by decanting the cells and wash three times in PBS by centrifugation (150 g for 10 min at 4°C). This technique does not alter cell viability, as assessed by the uptake of trypan blue.

potassium iodide and 3.7 × 10 6 Bq 125

4.9.4 Biosynthetic labelling of hybridoma-derived antibody

Secreted proteins, as well as cellular components, can be labelled biosynthetically by incorporat- ing labelled amino acids into a culture medium. Immunoglobulins, and many cell-surface and secreted molecules, are glycoproteins; therefore, radioactive sugars, as well as amino acids, can be used as labelled precursors. A culture medium deficient in the particular amino acid must be used to support cell culture during the incorporation of the labelled residue. Culture medium normally contains glucose as the sole sugar as cells biosynthesize the other sugars. If another sugar such as labelled galactose is added, little conversion to other sugars occurs. To optimize the utilization of the labelled sugar the level of glucose may be lowered, but cannot be omitted completely as cell viability and therefore incorporation of the label will be adversely affected.

C H A P T E R 4: Antibodies as probes

MATERIALS AND EQUIPMENT Hybridoma cells (or other cells in tissue culture) Horse or fetal bovine serum, dialysed against phosphate-buffered saline (PBS)

Selective culture medium deficient in leucine (selectamine is ideal) containing either 3 H-leucine (74–740 × 10 3 Bq/ml) or 14 C-leucine (37–185 × 10 3 Bq/ml), and/or 14 C-galactose (37–185 × 10 3 Bq/ml), in each case with 5% dialysed serum Plastic culture tubes, sterile

METHOD

1 Count the cell suspension.

2 Add 2 × 10 5 cells to a sterile culture tube and centrifuge at 150 g for 10 min at room temperature.

3 Remove the supernatant and add 0.2 ml of the labelling medium.

4 Incubate overnight at 37°C in a humid incubator gassed with 5% CO 2 in air.

5 After incubation, centrifuge (150 g for 10 min at room temperature) and remove the supernatant.

6 Determine the protein-bound radioactivity and specific activity.

7 Store at –20°C. TECHNICAL NOTES

• 14 C-leucine gives a higher energy emission than 3 H-leucine and so is counted more efficiently in

a scintillation counter, but 14 C-amino acids are produced at a lower specific activity than the 3 H form and they are more expensive. • 14 C and 3 H have far longer half-lives than 125 I; therefore, antibodies labelled with these radioiso- topes have a longer potential lifetime for use.

• 35 S-methionine is another amino acid frequently used for biosynthetic labelling of proteins. It is

available at very high specific activity and has a higher energy of emission than 14 C or 3 H, but a

shorter half-life. This amino acid is less frequent in the average protein, about one in 20 residues, so the protein is usually labelled to a lesser specific activity. The higher energy of emission is an advantage when this label is used in fluorography (see below in Section 4.10). Seleno-methionine

( 75 Se-methionine) is a g emitter and so is very easy to detect, especially in whole-body systems. It has the significant disadvantage that the radioactive moiety may be cleaved in vivo and so is not always a reliable tracer for the original amino acid.

• Sufficient incorporation of labelled precursor may be achieved by incubation times of only 3–4 h. This may be necessary if the tissue culture conditions are not ideal for cell maintenance. • Tissue culture media prepared for biosynthetic labelling by the omission of an amino acid should not be used for the determination of the rate of protein synthesis by incorporation of isotopically labelled amino acids, as the rate of uptake and incorporation is often crucially dependent on the external concentration of amino acids.

• Biosynthetic labelling in vivo may be achieved by injecting mice intraperitoneally with

1.85 × 10 7 Bq of a 3 H-labelled amino acid mixture: inject once daily on days 7–10 after the

4.9RADIOLABELLING OF SOLUBLE PROTEINS

be approximately 3.7 GBq/mmol. Remember that the mice will excrete 3 H after day 7, so ap- propriate precautions to contain the radioactivity and prevent personal contamination must be taken.

4.9.5 Conjugation labelling

Most proteins can be labelled easily by one of the direct methods, but some antibodies, particu- larly those with many tyrosines within the combining site, can be damaged by these methods.

Bolton and Hunter designed a compound, N-succinimidyl-3-(4-hydroxyphenyl) propionate, which can be labelled easily by the chloramine T method (see Bolton & Hunter 1973). The labelled ester is isolated by gel filtration and then mixed with the protein so that conjugation occurs via an amide linkage to lysine residues on the protein. The ester (Bolton and Hunter reagent) is also available commercially ready labelled with 125 I.