1. Recently we did transfection of our gene of interest (cloned in pEGFP-C1 vector) in HeLa cell, and we found that only few cells were transfected and those few cells show a diffuse localization through nucleus (please refer to the picture).
2. We are suspecting that our protein forms inclusion body in nucleus, please do comment on this and what alternative strategies can be done so that it will enable us to study the function and localization of this protein in the mammalian cell.
3. The protein which we are interested is uncharacterized, even its localization is not studied till date.
do some control experiment with the empty vector. CMV promoter plasmids always generate this kind of response (or similar, the picture is not too detailed to see...). in control, you should .reach 40-60% efficiency of transfection. than do again with your new target and use higher resolution with DIC preferably. this looks phase-contrast with low illumination, which might cover low fluo signal that probably exists.
hi
1- you need to repeat as transfection process with empty vector as control .
2- Your transfection rate is very low and you can prove that the unknown protein localization by cloning in pEGFP-N1 vector .It is also of note because a lot of light microscopy of the EGFP emits almost seems this protein in all cells, even in transfected cell culture medium .
good luck
yes, you are using the wrong delivery vector, which apparently comprises the cell's structure. we use LDL and see expression only outside the nucleus. the pic suggests dead cell debris.
Following simple experiment will give you answer, whether protein has expressed as a soluble form or inclusion bodies.
Harvest transfected cell and lyse them (preferably non chemical method). After lysing spin it down to separate supernatant and pellet. Dissolve pellet and run both fraction on SDS-PAGE to look for the protein of interest (It would be easy to identify if it is over-expressed, otherwise result may not be obvious or if possible try western blotting). Please dont use guanidine to dissolve the pellet, it will give you trouble in SDS-PAGE. Following papers may help you. Furthermore, if it is a membrane protein, interpretation may not be simple.
http://www.ncbi.nlm.nih.gov/pubmed/16233795
I hope this will help you.
Nilesh
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