Dear community,

After repeated defrosting of my samples, western blot control protein amounts were repeatedly irregular. I repeated the Bradford assay two times. Proteins were accurately diluted and accurately loaded into the gel. Still bands showed different amounts of protein in the repetitions. Gels did not overflow during loading.

We use protease inhibitors, proteins were denatured at 95 °C for 5 min before loading.

We know that GAPDH can be regulated during different conditions, e.g. hypoxia. This is not the case for the experiments we are currently running.

Any helps and hints are appreciated!

Best