By Alexandra C. Newton
The 1st compilation of vintage and rising laboratory thoughts for the learn of the biochemistry and phone biology of protein kinase C (PKC). defined in step by step element, those tools might be simply used to discover the constitution, functionality, rules, subcellular localization, and macromolecular interactions of protein kinase C.. every one protocol is brought within the context of PKC functionality and rules and comprises many notes on how most sensible to accommodate the issues that could happen. entire and authoritative, Protein Kinase C Protocols is a well timed compilation of biophysical, biochemical, cellphone organic, and molecular organic methods that brings protein kinase C study into any laboratory attracted to learning it.
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The 1st compilation of vintage and rising laboratory suggestions for the learn of the biochemistry and mobilephone biology of protein kinase C (PKC). defined in step by step element, those equipment will be simply used to discover the constitution, functionality, rules, subcellular localization, and macromolecular interactions of protein kinase C.
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Additional resources for Protein Kinase C Protocols (Methods in Molecular Biology Vol 233)
2. Chromatography on Mono-Q-Sepharose 1. 7 mL) is equilibrated with 10 mL of BQ. 2. The combined fractions BN50 and BN100 of the Ni-NTA chromatography are diluted 1:5 with BQ and loaded onto the column. 40 Gschwendt and Stempka 3. The column is washed with 10 mL of BQ. 4. Then the kinase is eluted with 1 mL each of BQ200, 300, 400, and 500. 10% glycerol and 10 mM β-mercaptoethanol are added to the eluates immediately. His-PKCδ is detected in the fractions and its kinase activity is measured as described above for the Ni-NTA chromatography.
11. Load sample onto an empty column and wash with 30 column volumes or more of buffer A. 12. Elute the recombinant protein four times with 100 µL of buffer B in each tube (see Note 11). 13. Dialyze the eluate to remove glutathione when necessary, and store the enzyme as described above. 7. 1. SDS-Polyacrylamide Gel Electrophoresis (PAGE) 1. 05 and 50%, and the theoretical maximum protein yield is 10 µg to 10 mg per 107 cells. For PKCs and PKNs, we usually obtained several µg to 100 µg of the purified enzyme from 107 cells.
1. Single-Affinity Purification of GST-Tagged PKC and PKN (Starting from a 5 × 10-cm Dish) 1. Thaw the cell pellet. 2. Resuspend cell pellet with 4 mL of buffer A with 1 mM phenylmethylsulfonyl fluoride. and incubate on ice for 10 min. 3. Homogenize using Dounce homogenizer with 30 strokes 4. Add Triton X-100 (final concentration = 1%; see Note 7). 5. Incubate at 4°C for 10 min 6. Centrifuge at 100,000g for 30 min and collect supernatant. 7. Add 400 µL of 50% slurry of glutathione Sepharose 4B (Pharmacia) equilibrated with buffer A to the lysate, and rotate at 4°C for 30 min.