PC3 cells stably expressing FLAG-tagged EphA2 were lysed in lysis buffer without phosphatase inhibitors and used to immunoprecipitate EphA2 with 40 μl of FLAG M2 affinity gel. Beads were washed twice with 1 ml of kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.1 mM EDTA, 2 mM dithiothreitol [DTT], 0.01% Brij 35) and aliquoted in four tubes. Each aliquot was resuspended in 50 μl of kinase buffer with 1) no ATP or kinase (unpublished data), 2) 200 μM ATP (9804; Cell Signaling Technology) and no kinase, 3) ATP and CK1 kinase, and 4) ATP and PKA kinase. Immunoprecipitates were incubated for 30 min in an orbital shaker (300 rpm) at 30°C. Kinase reactions were stopped by the addition of SDS-containing sample buffer, followed by incubation at 65°C for 15 min.
Atp 9804
Manufactured by Cell Signaling Technology
ATP (9804) is a laboratory reagent provided by Cell Signaling Technology. It serves as a source of the biological molecule adenosine triphosphate (ATP), which is essential for various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using atp 9804
1
Probing EphA2 Kinase Activity by Immunoprecipitation
2
Kinase Assay of TGF-β1 and PRKACA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The in vitro kinase assay was performed as described previously51 (link). Briefly, Flag-TGF-β1WT or Flag-TGF-β1T282A was transiently overexpressed with Myc-PRKACA in HEK293T cells and purified using anti-Flag magnetic beads or anti-Myc magnetic beads (88842, Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s protocol. The precipitated Flag-TGF-β1, Flag-TGF-β1T282A, and Myc-PRKACA proteins were resuspended in 40 μl of 1 × kinase buffer (9802) supplemented with 200 μM ATP (9804) (Cell Signaling). The reaction was carried out for 30 min at 30 °C and was terminated by the addition of 20 μl 3 × SDS sample buffer. Each sample was then boiled for 10 min at 100 °C and subjected to SDS–PAGE and IB analysis using the indicated antibodies.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!