have you made a new plasmid preparation in between?

Hi Daniel!

I always run both cell lysate and the cell supernatant samples (20ul) on my WB. Thanks for your suggestion on the boiling, it's worth a shot.

 Elitsa,

We have verified that our cells are in fact receiving our plasmid.  We have collected transfected cells, lysed them open, and conducted a PCR and digest to verify our plasmid is there.

Hey Daniel,

I would suggest to sequence the plasmid, to make sure, that what you are putting to your cells is in frame and correct. Also when loading the samples from the beads always use an input to see if protein is there (You can also use another flag tagged protein that is working well as a positive control). As for loading your pull-downs on the gel, you can resuspend your washed M2 beads with 2xSDS loading buffer and boil it for 10 min, then spin down the beads and load on the gel.

Good Luck!

hi Gina,

you plasmid might be detectable in the cells but just one point mutation within it can result in an unexpected stop codon and thus in non-fully (non-functional) expressed protein. Is your FLAG-tag N- or C-terminal? I think it will be faster and less frustrating if you sequence your plasmid before troubleshooting the western blot.

Good luck!