Covalent coupling of proteins to latex beads
| Materials | |||
|---|---|---|---|
| Borate buffer | 0.2 M of boric acid with 1 M NaOH, adjusted to a pH value of 8.5 | ||
| 2% Carbodiimide solution | 2% 1-(3-Dimethylaminopropy)-3-ethyl-carbodiimide hydrochloride are dissolved in MES buffer. Prepare only 15 minutes before use | ||
| Carbonate buffer | 0.1 M Na2CO3 are added to 0.1 M NaHCO3 until a pH value of 9.6 is reached | ||
| MES buffer | 0.1 M MES in water, pH value of 5.2-6 | ||
| Storage buffer | 0.01 M NaH2PO4 (pH 7.4), 1% BSA, 0.1% NaN3, 5% glycerol |
Procedure
200 μL 2.5% solution of carboxylated polybead-microspheres (latex beads) were mixed in a 2 mL Eppendorf tube with 1 mL of carbonate buffer and centrifuged for 6 minutes at 10,000 rpm. The supernatant is discarded and the latex beads are resuspended in 1 mL of carbonate buffer and washed again. Then the latex beads are washed four times with 1 mL of MES buffer. After the fourth washing step the beads are resuspended in 0.625 ml MES buffer. 0.625 mL of a freshly prepared 2% carbodiimide solution are added dropwise to the beads. The latex beads incubated at a maximum of 4 h, on a rotor at room temperature under inverting. After this time, the latex beads are centrifuged, the supernatant is discarded and washed three times with 1 mL MES buffer. Thereafter, the latex beads are resuspended in 600 μL of borate buffer and 200-400 μg of protein to be coupled are added. The beads are incubated in the rotor overnight at room temperature.
The next day, 25 μL of ethanolamine solution are added and the bead solution is inverted for 30 minutes in the rotor. This step is done to block free reactive groups on the surface of the latex beads. Thereafter, the latex beads are centrifuged for 10 min, the pellet is resuspended in 1 mL of borate buffer with 1 mg/mL BSA and incubated for 30 minutes at room temperature. The latex beads are washed once with 1 mg/mL BSA in borate buffer and then resuspended in 200 µL of storage buffer.
To control coupling, samples may taken during the working steps (each 30 μl): immediately after the addition of the proteins, before and after the addition of the ethanolamine solution, and after the first and second washing with borate buffer + 1 mg/mL BSA. Thus, the coupling of the proteins and the possibility of further losses can be verified by SDS-PAGE and Western blot.