After being treated with culture medium or EBSS (starvation) for 1 h, MEFs transfected with the indicated plasmids were harvested. Cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1% TritonX-100, protease inhibitor cocktail, pH 7.4) and immunoprecipitation with anti-FLAG antibody was performed as described above. Immunoprecipitates were split into two equal parts, one for a loading control and the other for in vitro kinase assay. Immunoprecipitates were washed three times in washing buffer containing 50 mM Tris-HCl and 500 mM LiCl, pH 7.4, followed by washing for three times in a reaction buffer containing 10 mM Tris-HCl, 100 mM NaCl and 1 mM EDTA. Immunoprecipitates were then resuspended in 50 μl reaction buffer and 15 mM MnCl2 before 20 μg sonicated phosphatidylinositol was added. The reaction was performed in the presence of 50 μM ATP and stopped by the addition of 1.5 M HCl. Organic phase was extracted with 200 μl chloroform:methanol (v/v, 1:1) and resolved by TLC using a coated silica gel in a solvent comprising chloroform:methanol:H2O:ammonium hydroxide (v/v/v/v, 9:7:1.7:0.3). The gel was imaged by Typhoon 9400 Variable Imager (GE Healthcare, Typhoon 9400).
Typhoon 9400 variable imager
Manufactured by GE Healthcare
Sourced in United Kingdom
The Typhoon 9400 Variable Imager is a laboratory equipment used for imaging and analyzing fluorescent and chemiluminescent samples. It is capable of detecting and quantifying a wide range of fluorescent and chemiluminescent signals in a variety of sample types, including gels, membranes, microplates, and petri dishes.
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5 protocols using typhoon 9400 variable imager
1
In Vitro Kinase Assay of Immunoprecipitated Proteins
2
In vitro Vps34 Lipid Kinase Assay
3
Kinase Assay for Autophagy Regulation
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Immunoprecipitation was performed with 500 μg protein from post-nuclear supernatant, using antibodies against ATG14 (MBL) overnight at 4 ° C. Samples were incubated with 30 μl Dynabeads (Life Technologies) for 1– 2 h at 4 ° C and then washed three times with lysis buffer. Two thirds of the beads were reserved for western blotting, while the remaining third was used for the kinase assay. Beads for the kinase assay were washed once with wash buffer (20 mM HEPES pH 7.4, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, and 0.05 mM DTT) and then resuspended to a final volume of 50 μl with reaction buffer (20 mM HEPES pH 7.4, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, and 0.05 mM DTT, 50 mM cold ATP, 5 mM MnCl2, 0.1 mg/ml sonicated phosphatidylinositol). 10 nM wortmannin was included in the indicated controls. The reaction was started with the addition of γ-32P-ATP (5 mCi) and the samples were shaken at 37 ° C for 30 min. Reactions were stopped with 120 μl stop buffer (CHCl3/CH3OH/HCl at a 10:20:0.2 volume ratio) and then shaken for another 10 min at room temperature. Samples were centrifuged for 5 min at 1000 g. 15 μl of the lower, organic phase was resolved on silica coated TLC plate (Millipore) using CHCl3/CH3OH/NH4OH/H2O (86:76:10:14 volume ratio) and visualized with the Typhoon 9400 Variable Imager (GE Healthcare Biosciences).
4
In vitro Vps34 Lipid Kinase Assay
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In vitro Vps34 lipid kinase assay was performed as previously described with minor modification18 (link). Briefly, Vps34 immune-complex beads were washed once with kinase assay (KA) buffer (20 mM HEPES, pH7.4, 1 mM EGTA, 0.4 mM EDTA and 5 mM MgCl2), then suspended in 50 μl KA buffer containing 50 μM cold ATP, 5 mM MnCl2, 50 μM DTT, 0.1 mg/ml Sonicated phosphatidylinositol and 5 μCi 32P-ATP. The mixtures were incubated at 37°C for 30 minutes with vigorous shaking. The reactions were stopped by adding 120 μl of CHCl3/CH3OH/HCl (10:20:0.2) followed by vigorous shaking for 5 min. The organic phase (lower phase) was separated by centrifuging for 5 min and 15 μl of the organic phase was spotted onto a silica gel thin-layer plate followed by chromatography separation for 30 minutes in CHCl3/CH3OH/NH4OH/H2O (86:76:10:14). 32P-PI(3)P radioactivity was recorded with Typhoon 9400 Variable Imager (GE Healthcare Biosciences, Little Chalfont, UK).
5
In Vitro Kinase Assay of ULK1
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Myc-ULK1 WT, myc-ULK1 kinase inhibited, or myc protein was immunopurified from HEK cells using anti-myc antibodies. IPs were washed once with kinase buffer (20 mM HEPES pH 7.4, 12.5 mM beta-glycerophosphate, 25 mM MgCl2, 5 mM EGTA, 0.25 mM DTT, 100 μM cold ATP, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche)) and then resuspended to a final volume of 50 μl with reaction buffer including 5 μg of substrate (ATG14 a.a. 20–73 or MBP) and 5 μCi 32P-ATP. Reaction was incubated at 37 °C for 30 min. Samples were subject to gel electrophoresis in a 4–12% bis-tris gel. The gel was visualized with the Typhoon 9400 Variable Imager (GE Healthcare Biosciences).
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