This method requires that a minimum of 0.5 mg of homogenous soluble purified protein has already been prepared (see Note 1). The purification procedures for the catalytic domain of PTP1B followed those previously described with the exception that Buffer B in Table 1 contained 25 mM NaH.PO. (12).

2.1. Crystallization of PTP1B After Oxidation in Solution

2.1.1. Oxidation of PTP1B in Solution

1. A minimum of 0.5 mg of purified PTP1B or 20 pg of purified protein for each crystallization condition that will be tested (see Note 1).

2. (Optional) p-NPP assay buffer: 10 mMTris-HCl, pH 7.5; 25 mM sodium chloride; 0.2 mM ethylenediamine tetraacetic acid (EDTA; see Note 2).

3. (Optional) 200 mM para-nitrophenyl phosphate (p-NPP). The solution can be stored at -20°C.

4. Hydrogen peroxide (purchased as a 30% solution; see Note 3).

5. Dialysis tubing and clips.

6. Oxidized PTP1B crystallization buffer: 4 L of 10 mM Tris-HCl, pH 7.5; 25 mM sodium chloride; and 0.2 mM ethylene-diamine tetraacetic acid (EDTA) (see Note 2).

7. 0.2-pM filters for degassing buffers.

8. Bradford assay reagent for determining protein concentrations.

2.1.2. Crystallization of PTP1B That Has Been Oxidized in Solution

1. Purified concentrated oxidized PTP1B prepared as described in Section 3.1.1.

2. Sterile 50-mL containers for storing crystallization buffers (Falcon tubes, for example).

3. Crystallization stock solutions (prepare 50 mL of each solution and store at room temperature):

a. 1 M N-(2-hydroxyethyl)piperazine-W -2-ethanesulfonic acid (HEPES), pH 7.0.

e. 2 M magnesium acetate (MgOAc).

4. 0.2-pm filter for syringes.

6. Pre-greased Linbro 24-well plate with cover.

7. 22-mm silanized glass coverslips (enough for every crystallization condition that will be tested and extras in case they break; Hampton Research or Molecular Dimensions Limited).

2.2. In-Crystal Oxidation and Reduction of PTP1B

2.2.1. Crystallization of Reduced PTP1B

Was this article helpful?

0 0

Post a comment