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.
Pak5 kinase
Manufactured by Cell Signaling Technology
PAK5 Kinase is a protein kinase that belongs to the p21-activated kinase (PAK) family. It is responsible for the phosphorylation and regulation of various cellular substrates, playing a role in cytoskeleton organization, cell motility, and survival signaling pathways.
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Lab products found in correlation
2 protocols using pak5 kinase
1
PAK5 Kinase Activity Assay
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.
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