Prepared by
Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111

Table of Contents


This technique is based on the polymerization of acrylamide with N,N'-methylene-bis-acrylamide (Bis) as the cross-linking agent. The degree of cross-linking, thus, can be partly controlled by adjusting the ratio of acrylamide to Bis used.

List of Reagents and Instruments

A. Equipment

B. Reagents


  1. Buffered Monomer Solution: Add 1.1 g of Bis and 20 g of acrylamide to a 100 ml of buffered solution (pH 7.0) of 0.1mM EDTA and 0.1M Tris-HCl in a beaker. See Note 1.
  2. To 10 ml of the buffered monomer solution of the above step, add enzyme powders (approximately 0.1ml of 75g/l fungal amylase) or an equivalent concentrated enzyme solution; mix.
  3. For 20 minutes, purge the dissolved oxygen in the solution that can interfere with the polymerization process with nitrogen. This step is critical in achieving a high degree of cross-linking.
  4. Add 0.1 ml of dimethylaminopropionitrile; mix.
  5. Add 1.0 ml of freshly prepared 10g/l potassium persulphate solution to initiate polymerization.
  6. Now is the time to pour the solution into a mold if one does not desire the gel to form in the original beaker. Leave the solution undisturbed; gel will form in approximately 10-30 minutes. (Hardening can be accelarated by using more dimethylaminopropionitrile.
  7. Cut the resulting gel into small cubes of approximately 3mm per side. Alternatively, if smaller pieces are desired, the gel can be forced through a syringe fitted with a fine needle.
  8. Gently wash the free enzyme off the gel surface in 10 ml of the Washing Solution. Repeat the washing process two additional times.


  1. The pH of the buffer should be adjusted to match the optimum value of the enzyme to be entrapped.


The above methods of enzyme immobilization by gel entrapment can be directly applied to live cells with minor modifications. For example, dimethylaminopropionitrile used in forming the polyacrylamide gel may not be employed because of its toxicity to viable cells. The monomers of acrylamide are also somewhat toxic to cells. On the other hand, cells can be immobilized with much less degree of cross-linking due to its much larger size.


  1. M.D. Trevan and S. Grover, Trans. Biochem. Soc., 7, 28, 1979.

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Enzyme Entrapment in Polyacrylamide Gel
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