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Procedure for modification of the ligand with PDEA

Under these conditions, PDEA reacts with carboxyl groups on the ligand. In addition to introducing a reactive disulfide into the ligand, this has the effect of raising the isoelectric point which can be an advantage in immobilization of acidic proteins.

Materials

0.1 M morpholino-ethanesulfonic acid (MES) buffer pH 5.0]
15 mg/ml PDEA in MES buffer
0.4 M EDC in water
100 mM DTE in water

Procedure

Follow the steps below to modify a protein ligand with PDEA. Volumes can be scaled down if required.

1. Prepare a solution of 0.5 mg ligand in 0.5 ml 0.1 M MES buffer pH 5.0.
2. Add 0.25 ml 15 mg/ml PDEA in 0.1 M MES buffer pH 5.0 (final PDEA concentration in the mixture 21 mM).
3. Add 25 μl 0.4 M EDC (final EDC concentration 13 mM).
4. Mix and incubate for 10 minutes at 25°C or 1 hour on ice.
5. Remove the excess reagents on a buffer exchange device equilibrated with a suitable buffer.

Determining the degree of modification

Aim for an average modification level of 1 substitution/protein molecule. The modification level can be controlled by adjusting the PDEA concentration, incubation time and temperature. If required, the degree of modification with PDEA can be determined approximately by reduction of the disulfide bond and spectrophotometric estimation of the thiopyridone released (absorbance maximum 343 nm).

1. Measure the absorbance of the modified protein at 280 nm (A280) and 343 nm (A1343).
2. To 1 ml protein solution, add 50 μl 100 mM DTE in water. Mix and allow to react for a few minutes at room temperature.
3. Measure the absorbance again at 343 nm (A2343). Calculate the degree of modification as follows:

See also

List of help files