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reporter gene -seap - expression of seap is very low (Jul/07/2007 )

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I am trying to transfect 3 plasmids together in hela cells using lipo2000 as a part of prote-nprotein intercation study.

I am using seap assay (clontech) to determine the transfection , but the level of seap is just 3-4 folds compared to un transfected cells (which tells very low transfection ).

please help and any suggestion are welcome. thanks!

-jhilmil-

QUOTE (jhilmil @ Jul 7 2007, 04:01 PM)
I am trying to transfect 3 plasmids together in hela cells using lipo2000 as a part of prote-nprotein intercation study.

I am using seap assay (clontech) to determine the transfection , but the level of seap is just 3-4 folds compared to un transfected cells (which tells very low transfection ).

please help and any suggestion are welcome. thanks!



Did you have a control for transfection to know that it worked. Best would be with a fluorescent protien. This would give a better idea of transfection efficiency.

I am assuming you have optimized the conditions for transfection with the Hela cells.

-scolix-

yes i did try using gfp, the T-efficiency was ~50%, i am following invitrogen instructions..the cells r nicely adehered after 48-55 h.

so i am assuming that my tranfection is working.

i am guessing my transfection is working, the 10%fbs conc is giving high seap level in untranfected cells.

i dont know ho to reduce fbc conc and still transfect my cells.
thanks

-jhilmil-

I thought the Hela cells could be transfected with a higher efficiencies. I could be very well mistaken.

I know when we used calcium phosphate transfection we would reduce FBS conc to 2% and transfect. Later we would add FBS after 12 hrs.

I havnt tried transfecting Hela cells with calcium phosphate.

-scolix-

when i do my transfection, i plate hela cells in 10% fbs a day before without p/s and on day on tranfection i use media (opti-mem) without phenol-red, fbs, and p/s and after 6 hrs, i change the media to complete media.

if i am correct, hela cells divide in 19-20 hrs , am i right..??

...so i am assuming the dna should be introduced around that time for maximal transfection efficiency, do u take any other precaution while transfecting ?
thanks!

-jhilmil-

did u do a heat treatment to kill the bgd seap activity from fbs? u can switch to luciferase system to avoid the background problem.

-genehunter-1-

QUOTE (genehunter-1 @ Jul 8 2007, 10:24 AM)
did u do a heat treatment to kill the bgd seap activity from fbs? u can switch to luciferase system to avoid the background problem.


ya since this seap enzyme is secreted in the medium, i just take 100ul and spin down to remove dead cells and then heat at 65 deg for 45min to heat inactivate.

is there a possibility that if transfection efficiency increses say to 80-90 %, this bgd will become insignificant?

pls suggest.
thanks

-jhilmil-

When did you collect the medium? How many hour after the changing of medium? What is the ratiio between the three constructs? What are the promoters of the other vectors?

SEAP is under SV40 promoter. The expression will be considerably lower compared to CMV. If you only use it as indicator and use very little SEAP plasmid also, of course you will not have much secreted protein. Another point is that the secreted level of SEAP will reach the peak only after 48-72h. Take care of the reading time also, when you do the assay. If it is just for transfection control, either fluorescent reporter or if you don't need to keep cells growing, use luciferase, much more sensitive.

-Almasy-

For assay system, SEAP has a higher background than luciferase system does. I used CMV-SEAP before and got 10-100 fold over background, if I remember correctly.

Follow Almasy's suggestions and see what happens.

-genehunter-1-

QUOTE (genehunter-1 @ Jul 10 2007, 11:53 AM)
For assay system, SEAP has a higher background than luciferase system does. I used CMV-SEAP before and got 10-100 fold over background, if I remember correctly.

Follow Almasy's suggestions and see what happens.


Thanks guys,

Almasy's-----My two plasmids are under SV 40, which directs the expression of seap. and seap is a reporter, For 24 well plate, i take 0.3 ug of each plasmid dna.

I did collect the sample after 50 hrs , my problem is seap level is never more than 5-10 folds compared to untransfected cells.

i cant use luciferase system as we r doing protein-protein interaction and clontect has now shifted to seap reporter.

i heat my samples at >65 for >45 min, but still no luck !

I would appreciate any other suggestion/comments.

thanks

-jhilmil-

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