my protein is a nucleolar protein which is tagged with HA, FLAG and myc tag. i have over-expressed it and good GFP expression was observed which is directly proportional to the amount of my protein expressing. but when i am making nuclear extract out of these cells after 48h of infection with prominent gfp expression with protein concentration measured by Bradford assay was 10ug/ul followed by western blot, i am not getting any band on the blot. what could be the possible troubleshoot for this.
Looks like a transfer problem because the wm doesnt look nice either.
did you run a sds page for evaluate the protein profile prior to western?
i think you could be running too fast in too little time the western, you can add an icepack to the camera to low the temperature. and before blotting you can use phenol red to see if there is a right protein profile in the membrane. hope it help.
Hi, seems sample degradation or infection. Have you tried other samples such as tissues etc with the same method?!
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