I've been doing a RGS-10 Knockdown in BV2 cells treated with LPS, FBS, and Serum free media. I blot with PKA substrate antibody and am not able to achieve a smear of bands.
PKA pathway is primarily a downstream effector of the TORC1 signaling complex. Using a number of reporters for the PKA pathway In general, the in vitro phosphorylation assays are performed with HA epitope-tagged proteins that are under the control
of the yeast CUP1 promoter in the yeast strain, PHY1942. The strains are
grown to mid-log phase in selective SC minimal medium containing 2%
glucose, and induce with 100 M copper sulfate for 90 mins. The HA
epitope-tagged Atg13 variants are isolated on an -HA antibody resin
(Roche), and incubated with [-32P] ATP (PerkinElmer) and 5 U bovine PKA
catalytic subunit (Sigma), A Western immunoblot control is performed with an -HA antibody (Sigma) to assess the relative amount of Atg13 present in each sample.The Atg13 protein is used as a substrate for PKA.Protein extracts for Western
blotting are prepared by a glass bead lysis protocol.The resulting protein extracts is separated on SDS-polyacrylamide gels and transfer to nitrocellulose membranes (Hybond ECL, Amersham Biosciences) at 4 °C. The membranes is probed with the appropriate primary and secondary antibodies and the Supersignal chemiluminescent substrate (Pierce) is used to illuminate the reactive bands.
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