Materials

2.1. Single-Point Attachment: Thermal Unfolding

2.1.1. Activation of Silica Beads

1. Controlled pore carrier (CPC)-silica beads, aminopropyl derivative, pore size 500 Ä, 30-45 mesh (Fluka, www.sigmaaldrich.com), 10 g (dry weight; see Note 1).

2. Washing buffer: phosphate buffer 100 mM, pH 7.0.

3. 10% (v/v) Aqueous glutaraldehyde (di-aldehyde) solution.

2.1.2. Protein Immobilization

1. Protein: ribonuclease A (RNase, Sigma, Type XII A, molecular weight [MW] 13.700).

2. Immobilization buffer: 100 mMphosphate buffer, pH 7.0.

3. Glutaraldehyde activated CPC silica beads.

2.1.3. Number of Linkages Between Protein and Support

1. Protein: ribonuclease A RNase (Sigma, Type XII A).

2. Buffer: 100 mM sodium phosphate, pH 7.0.

3. Aqueous valeraldehyde (monoaldehyde) solution: 10% (v/v).

4. Solution of trinitrobenzenesulfonic acid, according to standard literature conditions (15).

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