Immobilizing an enzyme normally requires optimization to achieve maximum activity recovery. Although the standard procedure is very straightforward, do not hesitate to contact ChiralVision if recoveries are unexpectedly low.
Prewashing of beads (all the IMMO kits are ready for use and does not require prewashing)
For anionic and cationic Immobeads it is important to adjust the material to the correct pH to ensure enzyme binding. Stir up the beads in an adequate volume and adjust with concentrated sodium hydroxide or hydrochloric acid. Once the desired pH is obtained (we advise 6.5 – 7.5) wash with 3 bed volumes of demineralized water and remove the excess of water. Now the beads are ready for use
Immobilization
Use a proportion of up to 4 ml of enzyme solution (10 – 100 mg protein / ml) per 1 gram (dry weight) of Immobead. The pH used should be close to the optimal pH for the enzyme. A value between pH 5 and pH 8 is most optimal. For epoxide beads use a minimum pH of 6.0 to ensure reaction. The quantities can be either scaled down or up, as long as the ratio is maintained. Working with disposable 1.5 mL vials 100 mg beads and 400 μl enzyme is a convenient quantity for screening purposes. Mix beads and enzyme on an ice bath at 4°C. After mixing thoroughly with a spoon, let the beads stand overnight at 4°C in the fridge without stirring or shaking. On larger scale (100 g and above) stirring or shaking for a few hours may be needed prior to overnight incubation.
Aqueous work-up and storage
After overnight incubation check the supernatant to determine the degree of binding of the enzyme and wash the beads 5 times with 2 bed volumes of cold purified water. Filter off the liquid, the beads can stay sticky.
For prolonged storage it is recommended to store the beads as dried material by drying with a stream of dried air. If stored wet use either 51% (wt/wt) propanediol or glycerol or saturated ammonium sulphate. Care must be taken that the enzyme is not washed off by these additives (applies only to the non-covalently bound enzymes). Samples in propanediol or glycerol can be stored at -20 °C, all other samples must be stored at 4 °C. For filtration either a P1 glass filter or a 100 μm sieve can be used.
Work-up with organic solvents
After overnight incubation wash the beads on a filter 5 times with 2 bed volumes of cold purified water. Filter of the liquid and wash 5 times with 2 bed volume of cold acetone followed by washing with room temperature acetone. Dry with a stream of dry nitrogen / air until all solvents has evaporated. Instead of acetone also 2-propanol can be used.
Download immobilization instructions standard beads
Download immobilization instructions HIS-tag beads
Please also carefully read optimal usage instructions for immobilized enzymes:
Download usage instructions immobilized enzymes