Vice versa, GPER-sfGFP was predominant in the insoluble fraction, which we expected since it is a membrane protein. GFP was so strongly expressed in the positive control cells that is was even seen in the insoluble fraction. The positive and negative control have worked as expected. Unfortunately, the assay wasn’t successful for the xLHCGPRs due to a systematic error. Have some “small washing” steps before the actual 5-15 minutes washing steps: Just let a little bit of PBST into the case with the membrane and pour it out a couple of times until the solution that comes out is not “milky” anymore. Tip from the Supervisors: When washing the membrane with PBST for the first time. The two pictures are merged so the protein ladder seen next to the spots on the Western Blot. Then a bright field picture is taken to visualize the protein ladder. First, a Picture that allows detection of the light emitted by the HRP reaction is taken. The two solutions of the visualization kit are mixed and directly pipetted onto the membrane.
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