Download e-book for iPad: Chaperonin Protocols (Methods in Molecular Biology Vol 140) by Ch. Schneider

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5 L flasks) pre-equilibrated to 37°C are then inoculated with 5–10 mL of the fresh overnight culture. The OD600 is measured every 30 min, using the uninocculated 2x TY medium as a blank. 6, 1 mL/L of stock IPTG is added to the culture (final concentration 1 mM). The cultures are then further incubated until the cells stop growing (absorption at OD 600 reaches plateau (approx. 0 OD600). Usually it takes about 3–4 h, after which the cultures are placed on ice, ready for cell harvesting. 5. It is advisable to check induction of the GroES during fermentation.

7. 5 µL of each fraction on a 15% SDS polyacrylamide gel to determine which fractions contain Gp31 (molecular mass is about 12 kDa). Stain the proteins in the gel with Coomassie brilliant blue (see Note 3). 35 M NaCl. 8. , item 12) overnight at 4°C.

5. 3. Ampicillin stock: 50 mg/mL in H2O. 4. 1 M (238 mg/mL) Isopropyl `-D thiogalactopyranoside (IPTG) stock solution in H2O. 5. Lysozyme stock solution: 10 mg/mL. 6. 0. 7. 1 M DTT (dithiothreitol) in H2O: 154 mg/mL. 2. Buffers All buffers are freshly made, degassed, and filtered just prior to purification. We normally carry out the chromatographic steps at room temperature. Therefore, the buffers are not prechilled. However, if chromatography is to be carried out at 4°C, the buffers should be equilibrated at this temperature.

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