Conventional Cross-linking Using Glutaraldehyde

Protein cross-linking with glutaraldehyde

Procedures

1. Check the protein buffer. Phosphate buffers at pH 7.5-8.0 and HEPES buffers are appropriate, whereas Tris-Cl buffer should be avoided.

2. 50-100 mg of proteins in 100 $\mu$L 20 mM HEPES (pH 7.5) buffer are treated with 5 $\mu$L of 2.3% freshly prepared glutaraldehyde solution for 10 minutes on ice. Chiou et al. (2006) suggested that the added amount of glutaraldehyde is calculated as equal moles of cross-linker reactive groups.
3. The reaction is terminated by the addition of 10 $\mu$L of 1 M Tris-Cl (pH 8.0).
4. The cross-linked sample will be characterized using SDS-PAGE, SEC, and negative-stain EM.

Notes

The crosslinkers need to react with proteins free from amines.