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

Hmm, this is a bit problematic. Now, first of all, I am not sure that Hela is a good cell line for SEAP. According to the Clontech booklet, they don't recommend this cell line due to high background. I understand that we are reasoning that with high transfection efficiency this theoretically should not be a problem, but still, since you are having problem with background (just like their warning), this may need to be taken into consideration when you design exps.

Second, is there any reason why you have to use SEAP? Can't GFP work, too?
Third, I understand that you need to study pro-pro interaction. So I assume that you transfect 3 constructs into the cells, 2 of them for the interaction part and the other is SEAP for checking transfection efficiency? If so, why can't you use luciferase? You still have to lyze cell to do whatever exp for Pro interaction, right? Maybe I haven't been able to get the point here, if so I am sorry, just ignore this point.

---

i've done SEAP experiments (invivogen psiTEST) using 293 and HeLa cells.
I use heat inactivated serum, and the background is low in 2 of 3 of my controls.
For the third one the level is relatively not negligible but don't interers with my exp.
I check SEAP 48h after transfection and got good results in OD measurements, either in transfecting siRNAs or an other plasmid

---

I also use SEAP (Clontech, same as you jhilmil). But I transfect the pSEAP-Control into 293, and do not have problem with background (see attachment). What if your other two proteins affect its secretion? Have you tried with just SEAP and see how?

---

Thanks guys,

I have incubated FBS at 65 C (with mixing in between) to heat inactivate FBS and then added to my DMEM to make the final concentration of 10% FBS.

Is it what you do (fred 33) to heat inactivate serum and then used as complete medium ?

When i do the assay for detecting SEAP level, the samples collected after 50 hrs are incubated at at 65 C for 45 min, wont that be sufficient to inactivate endogenous SEAP from hela cells, if the heat-inactivated FBS was not used in making complete medium??

Another thing, even if Hela cells r not the 100% choice for seap assay (cos of high background), there r many published studies done with Hela cells... so that makes me feel that something is wrong in my things.

Almasy-----since i am doing p-p interaction, the 2 plasmids have the proteins which r suppose to interact and 3rd plasmid ( seap reporter) gets transcribed (incase the 2 proteins interact) and that s what we check directly in culture medium (without lysing the cells).

Greatly appreciate anybody's suggestions.

thanks!

---

that's close to what i do. 56° 30' with mixing, and on a thawed serum. you mean you heat al proteins at this temperature??? i think this procedure is the one that you may try to avoid.
Using of heatinactivated serum seems ok for a try.

---

I heat inactivated cell supernatant, rather than serum.

---

QUOTE

you mean you heat al proteins at this temperature??? i think this procedure is the one that you may try to avoid.
Using of heat inactivated serum seems ok for a try.

---------YES , this what i have been doing so far ---heat inactivating the cell SUP and this is as per the SEAP assay protocol (clontech). Do you mean to say that incubation at 65 d is what making seap inactive in all the experimental samples and thats why everything including +ve and -ve controls show the same level.

In my last sets of experiments, i had inactivated the serum but then i read somewhere that we can use straight FBS also. Now i am using medium with normal serum (without heat-inactivation)

Is using heat-incativated serum very crucial?

what will be the consequences if the medium has normal serum (not heat-inactivated).
Thanks for the suggestions.

---

SEAP is heat resistant while the endogenous AKP is not. This is the principle behind heat inactivation step. I think the transfection rate was low and that is what causes the problem. Have you optimize the transfection conditions for your cell type? Try to transfect cells with SEAP alone, in the amount of total plamsid DNA that you used before and see what SEAP level can you achieve. Depending on the level of SEAP you get with SEAP plasmid alone, you may have to increase the SEAP plasmid: test plasmid ratio in the transfection mixture.

---

there are residual phosphatase activity in the serum so background may be higher. But assuming it's heat inactivated and SEAP not, well that's probably no pb to use straight serum.
do you have a reporter gene on your plasmid ?
when i did SEAP assays, i did an analysis showing me the proportion of transfected cells... at least 30% is required for decent exp.

---

Thanks fred 33 and genehunter-1

Genehunter-1..thanks for clearing my biggest doubt.

Today i have setup a new experiment and i have taken care of all these points . I did optimize the transfection conditons and confirmed with GFP. hopefully, i will get good results from this one.

But inspite of using same conditions and keeping no of cells same (previously), my transfection efficiency differs a lot from experiment to experiment (45-75%).

Since the plasmid containing the seap is a reporter plasmid, i use another plasmid which carries seap downstream of the promoter and hence is good transfection control.'

thanks

---