SEAP assay - i have to do the assay today, please help (Jan/06/2006 )

i have transfected HeLa cells with my constructs along with a reporter gene (SEAP). And i have to check the levels of SEAP (secreted form of human placental alkaline phosphatase) activity in culture medium within 48-72 hrs of transfection using chemiluminescence .

My question is, today i 'll take out some culture medium and do the assay, if i have to do the assay after 72 hrs do I need to change the medium (as its 36hrs since i have changed the medium and its becoming yellowish)

and if I have to then do I need to store the culture medium and then add fresh medium ?

Another question is, if I add fresh medium, will the cells keep secreting SEAP and that means i can do the assay again after 72 hrs and need not to store the culture medium ?

I think i am not understanding it well. Please advise.
Thanks ,
jhil

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hi
you need to differentiate two things :
if you want to test if at 72h your protein is still produced and excreted, you have to change medium and let sit the medium for the same time as the firste measures. Best is to do all assays at a defined hour for the three days. you will be able by this answer to answer the question and compare values.

second question : you need to study the half life of secreted protein : to see if medium is saturated you let the middle on the cells.

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thanks fred,

unfortunately, i need not understand your reply clearly.

today, i.e after 48 hrs of transfection, I did the SEAP chemiluminescence assay and it was a total disasster, my negative and positice construts showed the signal and all my samples were blank
(-ve). i dont know what to do next.

since my cells r still okk..i have not chnaged the medium todal aslo and am planning to change it on monday i.e 72 hrs. i will assay again on monday.

please suggest me if you have any suggestion.

thanks,
jhil

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positive and negative controls show signal ?.... there is sthg wrong in your exp as NEGATIVE is showing signal

i try to rephrase my post

first subject of your assay : see how long the protein is expressed and excreted by the cells :
you have to test a medium that was in contact with cells for a defined time. If you choose 24h :
Transfect day 1 and ensure transfection is completed at 14h. Day 2 at 14h : change medium (test the "24h" assay). Day 3 : change medieum at 14h test the "48h" medium.

The tested mediums were exposed to cells for the same time (24h each) and you will answer the question : my prot is excreted at 24, 48, and maybe 72h. As medium was exposed 24 for each condition, you will answer too the fact that , at 24h quiantity of prot was X, at 48h, quantity of protein excreted was y etc...


second subject : you need to study the half life of secreted protein :
you have to trasfect 3 batch of cells (d1), and test the first medium at day 2 (24h) and let the medium on plate/flask 2 and 3
day 3 : test the second batch and see the total activity
day 4 : test the third batch and see the total activity

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thanks fred, i really appreciate ur help and i have noted ur points.

i have another problem, i thk since my cells r nicely adhered. they look great under mscope even after 60 hrs and so

can i conclude that the transfection was okk (succesful)?

my second question, i am doing chemiluminescence to detect the expression of a reporter gene (SEAP) and when i tried detecting the very first time, i saw no signal although i was able to see color in all the wells.

since i got some signal in my negative control, i understand that i messed those somehow (although, i am quite sure but since results r saying something else, it seems i did some mistake).

there was a control to detect whether the detection system is working in our hands or not, the kit comes with a human placental alkaline phosphatase, which I added to the untransfected cells and added rest of the buffers and substrate. the sad part is, even this did not show any signal. (which makes me think that chemiluminescence is not working,,, but since i saw some signal with +ve /-ve controls,,,,,,i am confused))

the positive controls, which i had mentioned last time was as:

the kit has supplied two constructs which interact with each other and as a result the reported gene is transcribed and when i transfected those constructs, i got some signal.

the constructs, which do not interact with each other (negative control) , were also transfected in my experiment and ideally, there should not have been any signal but i did see some.

I doubt that chemiluminescence is not working with me. I have no experience with this detection method, maybe thats a part of reason i am not able to measure enzyme activity. I am using Typhoon to measure chemiluminescence in 96 well plate.

what are other ways to find out that the transfection did work with me (any visual test), apart from SEAP assay?

Thanks for your suggestions,
jhil

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