shaung

Hey, Having an issue with 293T cells and FGF stiumaltion assays. I grow 293T cells, transfect them with expression vectors containing proteins that i know increase response to FGF stimulation compared to untransfected cells, and i know that 293T cells endogenously express fgf receptor. i know the expression vectors express protein, via western blot, but the response to FGF stimulation after incubation in minimal media (krebs HEPES buffer) does not follow the expected outcome. when comparing even untransfected, non-fgf treated cells with fgf treated cells, no increase in pERK is seen (considered the measurable output for fgf stimulation), so the issue seems to be with the 293T cells or the Fgf, what do you guys think?? cells have been shown to be mycoplasma free.

i had a similar assignment during my honours year, and it really depends what you r interested in. personally, i am interested in cancer treatment options, so targetted cancerous cell treatment options was an area of interest for me, meaning you could look for unique membrane bound proteins to be expressed in cancerous cells, how to deliver the drug, side-effects of this form of this drug delivery method, etc.

are you running at the same voltage over the 7years? lower voltage may result in more passive diffusion across the lanes of the gel...

maybe compare beta actin levels with other house-keeping genes such as GAPDH.

ligation shouldnt be an issue if your are just excising a fragment, as long as you do the obvious, i.e. gel purification of the vector, otherwise you could just ligate the excised fragment back into it's original position.

not goin to answer for you, but: 1. what do you use to help visualise the proteins on a western and isnt used on a coomassie? I.e. what does your secondary antibody bind to in a western? why can you see so many bands in a whole cell lysate coomassie? 2. talk about native protein structure, binding partners, etc. 3. say you have a ball pit full of yellow balls (being your DNA template) and 1 green ball (being your primers) in a room with no gravity, how easy would it be for the green ball to move between the yellow balls to find their specific binding sites. hope this helps!