3 replies to this topic

[soymilk14]

I want to overexpress a transcription factor in a cell line. Can i use a plasmid fused with gfp to overexpress it? my question is, how can it enter the nucleus? and how can i verify its transfection efficiency?

thank  you

[laurequillo]

soymilk14, on Jun 30 2010, 05:42 PM, said:

I want to overexpress a transcription factor in a cell line. Can i use a plasmid fused with gfp to overexpress it? my question is, how can it enter the nucleus? and how can i verify its transfection efficiency?

thank  you

If the localization of your protein is nuclear, it will appear in the nucleus. You can check how many cells are green

[soymilk14]

thank you. can i use any plasmid? My protein of interest is a transcription factor.  I want it enter the nucleus. is an ordinary plasmid like pcDNA3 fused with gfp be enough to enter the nucleus and target the gene of interest? thank you.

[laurequillo]

soymilk14, on Jun 30 2010, 06:05 PM, said:

thank you. can i use any plasmid? My protein of interest is a transcription factor.  I want it enter the nucleus. is an ordinary plasmid like pcDNA3 fused with gfp be enough to enter the nucleus and target the gene of interest? thank you.

yeah sure, the localization of your protein is not affected by your plasmid. Sometimes GFP could affect the conformation of your protein, but normally it doesnt. To be sure you can create an Nterminal and a C-terminal GFP protein and compare both.

So if your protein has a NLS, is actively or pasively transported to the nucleus (the endogenous one) the one that you introduce with your plasmid should behave in the same way (sometimes you can see some diferences, but you should not see big differences)

Edited by laurequillo, 30 June 2010 - 08:52 AM.