Download Basic Cell Culture by Jeffrey W. Pollard, John M. Walker PDF

By Jeffrey W. Pollard, John M. Walker

ISBN-10: 0896033848

ISBN-13: 9780896033849

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Schmidt A, Hall A (2002) Guanine nucleotide exchange factors for Rho GTPases: turning on the switch. Genes Dev 16:1587–1609 29. Wolfman A, Macara IG (1990) A cytosolic protein catalyzes the release of GDP from p21ras. Science 248:67–69 30. West M, Kung HF, Kamata T (1990) A novel membrane factor stimulates guanine nucleotide exchange reaction of ras proteins. FEBS Lett 259:245–248 31. Yamamoto T, Kaibuchi K, Mizuno T et al (1990) Purification and characterization from bovine brain cytosol of proteins that regulate the GDP/GTP exchange reaction of smg p21s, ras p21-like GTP-binding proteins.

2 mg of Sulfo-SMCC crosslinker (Thermo Scientific). 6. Chemically synthesized HVR peptide (GKEKMSKDGKKK KKKSKC). 5 SortaseMediated Ligation 1. 15 μM sortase A (see Note 7). 2. 15 μM K-Ras catalytic domain stored at 4 °C. 3. Tris–EDTA buffer pH 9: 50 mM Tris–HCl, 10 mM EDTA, 10 mM MgCl2, 5 mM CaCl2 4. 10 mM βME. 5. 30 μM HVR peptide of K-Ras conjugated with S-farnesyl-Lcysteine methyl ester using Sulfo-SMCC crosslinker. 6 GTPase Activity Assay 1. 1 M GTP stored at −80 °C (see Note 8). 2. 10 μM fully modified K-Ras 4B stored at 4 °C.

1 Bacterial Expression 1. Place a single vial of one-shot BL21AI cells on ice and allow thawing. Add 1 μL of K-Ras 4B PET 42a plasmid to vial. Keep vial on ice for 30 min. 2. Heat the vial to 42 °C for 45 s and return to ice for a further 2 min. 3. After this, add 350 μL of sterile SOC medium to the cell vial and incubate with agitation at 37 °C and 250 rpm for 1 h. 4. Make a 350 mL solution of LB Broth supplemented with 350 μL of 50 mg/mL kanamycin solution. 22 μm pore-size membrane into a 1 L autoclaved conical flask.

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