Show description

3. Upon a heatshock at 42°C for 45 sec and cooling down on ice for 2 min, the bacteria are mixed with 400 µL of SOC medium and incubated at 37°C for 1 h. 4. 5% Bacto-Agar in LB medium in the presence of 50 µg/mL kanamycin and 30 µg/mL chloramphenicol. 5. The plates are dried for 10 min and incubated at 37°C for 12 h. 6. Bacterial colonies are transfered to 50 mL of LB medium containing the same antibiotics as above and incubated at 37°C for another 12 h. 7. 7. 8. 5 mM (final concentration) isopropyl1-thio-δ-D- galactopyranoside at 24°C for 12 h.

5, 10 mM β-mercaptoethanol. 13. Phosphatidyl serine liposomes: The commercially available solution of phosphatidyl serine (Sigma, Munich, Germany) is evaporated, buffer P is added (final concentration 1 mg/ml), and the suspension is sonicated on ice with a Branson sonifier (3 min pulse at setting 6). 14. Liquid scintillation cocktail, for example, Ready Safe (Beckman, Palo Alto, CA). 15. The pseudosubstrate-related peptide δ: MNRRGSIKQAKI. 16. Protein dye reagent concentrate (Bio-Rad, Munich, Germany).

These technologies offer greater flexibility and reduce the time it takes for further isolation of a single recombinant virus by plaque assay Expression and Purification of PKC from Insect Cells 23 because recombinant viral DNA can be checked before its introduction into insect cells. These methods are effective and have technical superiority to recombination. However, we usually use the recombination system in Sf9 cells for expressing various proteins, including isoforms of PKC and PKN, because many kinds of transfer vectors that can be easily manipulated and various virus DNA optimized for expression of foreign gene are now commercially available.