problem in transfection - (Apr/25/2010 )

hi
m new in transfection field. i try to transfect my vector plasmid 5+ 3 kb insert total 8kb plasmid into cho cells.i m unable to check wether my protein is transfected or not i also transfect egfp plasmid as contol and m able to see florousrence in control.
i tried to check with flow and blot but not able to see any positive result.

sequence of insert was confirmed by sequencing. i also check expression in dh5 strain , i used cell pellet and checked protein through blot but i m unable to see any protein band in blot ,antibodies were working because i used his taged positive control and use his tagg primary antibody as my protein is also his tag at c turminus.
can i check protein expression in dh5 strain because it is mammalian vector and t7 promoter?

is transfection efficiency matter with the size of vector test vector is 8 kb and egfp vector is 4 kb?

is lipofectamine+ plus reagent is good for large vector?

can i linerize my vector so that ther should not be any frame shift?

how the plasmid integrate in cell genome may be through homologous recombination?

no ...you can not use DH5a for protein expression with a T7 promoter. You need a DE3-lysogen like HMS174(DE3) or BL21(DE3).

Transformation efficiency is indeed influenced by vector size ...the smaller the better the transformation efficiencies. But this means not that your transfection efficiencies drop to zero compared with EGFP-vector that is half the size.

I would consider to check your vector once again for frame-shifts, mutations or things like that.

You are going for stable expression or transient?

Regards,
p

M also trying stable transfection side by side selection is there even after 3 weeks cho cells r growing very well in presence of .600mg /ml.

but even after selection my protein was not expressed on cell surface or in supernatant .

thanks

if you are able to raise antibiotic resistant clones ...and you are not able to detect you protein ...this is more or less an indication that there is something wrong with your expression unit.

This means that transfection was succesful ...the plasmid has integrated.
After what timespan do you tried to detect your protein? Maybe its already silenced?

Regards,
p

m trying to express my protein not silencing. clones r selected but they r not expressing at all as i checked by flow and blot. i want to express my protein on cell surface . please give me some suggestions to rule out this problem.

i checked protein after 72 hr of transient and 72 hrs of stable transfected clones.

thanks