The polymerisation and negative staining of Catalase

Catalase, from Beef liver, ’Sigma’, C-100, 25 mg/ml, 2 crystallized, aqueous crystalline suspension with 0.1 Thymol, pH 5.4.

Reagents:

0.1 M NaH2PO4 + 0.1 M Na2HPO4, pH 6.0

(NH4)2SO4 (’Merck’, No. 1.01217, 132.14 g/mol)

For the details see Kiselev N.A. et al, Crystallization of Catalase in the form of tubes with monomolecular walls, J.Mol.Biol., 1967, 25: 433-441.

 

The protocol:

Dissolve the required amount of Catalase (e.g. 40 l) in (60 l) 0.1 M sodium phosphate buffer (1 final of Catalase), add ammonium sulphate to obtain the final concentration of 1.6 M (21.14 mg; the buffer’s pH drops to 5.6), and leave in a fridge at +4C for 16 hr.

The final amount of Catalase in the solution has to be > 0.7.

Important! The Catalase powder from Bovine liver (’Sigma’, C 1345), does not polymerise under these experimental conditions!

  1. The prep (6 l) on the grid for 30 sec–1 min;

  2. wash with water (8 7 sec);

  3. stain with 1 uranylacetate.

 

2003 06 20 – Julius