BGC 823 cells were incubated with indicated concentration of small molecular compounds. Then cells were suspended in cold lysis buffer supplemented with protease and phosphatase inhibitors. Supernatants were preabsorbed with protein A-Sepharose 4B beads (GE Healthcare Bio-Science Inc., Sweden) for 1 h at 4 °C before incubation with PAK1 antibody (Santa Cruz). Then the supernatants with equal amounts of protein were subjected to immunoprecipitation with PAK1 antibody. MBP was used as substrates to assess PAK1 kinase activity. Kinase activity was measured in kinase buffer containing 10 μCi of [r-32P] ATP (5000 Ci mmol−1) for 30 min at 30 °C. Reactions were terminated by addition of 6 × SDS buffer and loading on a 12% SDS-PAGE. 32P-labelled proteins were transferred onto PVDF membranes and detected by autoradiography with Molecular Imager RX (BIO-RAD).
Molecular imager rx
Manufactured by Bio-Rad
The Molecular Imager RX is a laboratory equipment designed for the detection and quantification of radioactive or chemiluminescent signals in biological samples. It utilizes imaging technology to capture and analyze data from various experimental techniques, such as Western blotting, Northern blotting, and gel electrophoresis.
Automatically generated - may contain errors
4 protocols using molecular imager rx
1
Assessing PAK1 Kinase Activity
2
In Vitro Kinase Assay for PAK5 Activity
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GST-fusion proteins were purified in vitro and washed three times with kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 2 mM MnCl2 and 0.2 mM DTT). Commercialized PAK5 Kinase (Cell Signaling Technology Inc.) or immunoprecipitated cell synthesized PAK5 Kinase domain was used for kinase assay in 50 μl of kinase buffer added with 10 μCi of [γ-32P] ATP (5,000 Ci/mmol) and 2.5 μM cold ATP for 30 min at 30°C. Reactions were stopped by addition of 6 × SDS loading buffer. After 10% SDS-PAGE and transferred onto PVDF membranes, 32P-labelled proteins were visualized by autoradiography with Molecular Imager RX (BIO-RAD). Histone H3 (Invirogen) was used as positive control. Ponceau stain indicated the loading amounts of GST-fusion proteins.
3
PAK4 Kinase Activity Assay
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PAK4 kinase assays were performed using the exogenous MBP as substrate to assess activity. For chemical direct effects on PAK4 kinase activity in vitro, commercialized PAK4 kinase (Life) was pre-incubated with different concentrations of compound. Kinase reactions were started by adding MBP and a mixture of [γ-32P] ATP and 1 mM ATP. Kinase activity was measured in 40 μL of kinase buffer including 10 µCi of [γ-32P] ATP (5000 Ci/mmol) for 30 min at 30 °C. Reactions were stopped after mixing with 6× SDS buffer and loading on a 12% SDS-PAGE. Then, proteins were transferred onto PVDF membranes and proteins labeled with 32P were visualized by autoradiography with Molecular Imager RX (BIO-RAD). In order to guarantee the equal loading amount, PAK4 were detected by immunoblotting analysis and MBP (Life) was detected by Ponceau stain.
4
PAK5 Kinase Activity Assay
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The GST-fused proteins were expressed in BL-21 bacteria and purified with GST Sepharose beads (Amersham) and used for PAK5 kinase assay as described previously (10) (link). GST-fusion proteins were purified in vitro and washed three times with kinase buffer (50mM HEPES, pH 7.5, 10mM MgCl2, 2mM MnCl2 and 0.2mM DTT). And afterward for 60min at 30°C with commercialized PAK5 kinase (Cell Signaling Technology) or immunoprecipitated cell synthesized PAK5 kinase domain for kinase assay in 50μl of kinase buffer added with 10μCi of [γ-32 P] ATP (5,000 Ci/mM) and 2.5μM cold ATP. Reactions were stopped by the addition of a 6×SDS loading buffer. After 10% SDS-PAGE and transferred onto PVDF membranes 32 P-labeled proteins were visualized by radioautography with Molecular Imager RX (BIO-RAD). Histone H3 (Invitrogen) was used as a positive control. Ponceau stain indicated the loading amounts of GST-fusion proteins.
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