Thermo Fisher
GFP Tag Antibody, Molecular Probes
A-11122
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One of the goals of my project is to carry out protein-protein interactions with components that are relevant to the hedgehog signaling. A first step in the process is to test the antibodies to determine the expression in either over-expression or endogenous levels. Here, GFP antibody from Thermo Fisher was tested for immunoprecipitation of a GFP tag expressed in HEK293T cells.
Immunoprecipitation
GFP tagged protein over-expressed in HEK293T cells
For immunoprecipitation: 2 µg per 2 mg lysate, 1 hr incubation at 4 deg; For Western Blotting: 1:1000 dilution, 16 hr incubation at 4 deg
5% milk powder in 1x TBS-T (0.1%)
For Western Blotting: 1:10000 dilution of Donkey anti-Rabbit HRP antibody for 1 hr at room temperature
NA
ECL; exposure was 2 sec on a Film
GFP tag expressed in HEK293T cells was solubilized in RIPA lysis buffer. For immunoprecipitation, 2 mg of clarified extract was incubated with 2 µg GFP antibody and incubated for 1 hr at 4 deg on a rotator. 10 µl of Protein A Sepharose beads were added and incubated for 1 hr at 4 deg. Bound complexes were washed extensively and eluted in SDS sample buffer. For Western Blotting, the same antibody was used at 1:1000 dilution. The detection was specific.
None
Specific signal was observed
Good antibody for both immunoprecipitation and Western Blotting