Proteins are not directly immobilized to the substrate; spacer molecules can act as bridges connecting proteins to the substrate. Source: Laboratory Manual of Enzymology

Operation method

basic program

Materials and Instruments

Protein
Potassium dihydrogen phosphate NaCl N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride HCl

Move

For "Reagents" required for the experiment, see "Other".

The procedure for ligating spacers is the same as for ligating proteins (see "Binding of Proteins to Cyanogen Bromide-Activated Agarose") except that 1 mg/ml (20 mg/20 ml for the described procedure) of 1,6-hexanediamine is used in place of the protein. Washing off the hexyl derivative with 0.5 mol/L NaCl (AH-agarose)

To attach the protein to the spacer, adjust the pH to 4.5 with 1 mol/L HCl. Subsequently, add N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (10 mg/ml gel) and protein (10 mg/ml). The final pH must be maintained at 4.5. The mixture is shaken at 4°C overnight (pH adjusted after 1 h). Finally, rinse the gel with 0.1 mol/L pH 7.6 potassium dihydrogen phosphate.

Common Problems

Reagents:

0.1 mol/L pH 7.6 potassium dihydrogen phosphate

1,6-Hexanediamine (Mr = 116.2)

0.5 mol/L NaCl (Mr = 58.4; 2.92 g/100 ml)

N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl, Mr = 191.7)

1 mol/L HCl

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