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p53-luciferase activity


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#1 Thapa

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Posted 15 April 2009 - 05:41 AM

Dear all,
I have transfected p53-luc response element in HCT116 cells and observed change after drug treatment. In these experiments, I have found very small increase in luciferase activity (like 10-25% increase compare to vehicle treated control group). Please note that luciferase activity in control group is really very high.

In a previous western blot experiement, that particular drug had increased p53 level (protein) by 5 folds or higher.

What does that imply? I have just started new work and not have enough idea whether this two results should be consistent or not??

Warment regards,
Thapa
"Learning without thought is labor lost"

#2 dpo

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Posted 15 April 2009 - 08:13 AM

maybe your p53 stays in the cytoplasm when you treat the cells with the drug? you can check this by doing an immunofluorescence staining of p53 in the treated and non-treated cells

#3 bob1

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Posted 15 April 2009 - 04:47 PM

It implies that your transfection is not very good. 5 fold increase of endogenous protein may still be there, but the luciferase one is't being induced to the same level, therefore it is not working very well.

Note that luciferase experiments are fraught with difficulty and reproducibility issues.

#4 Thapa

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Posted 15 April 2009 - 06:36 PM

maybe your p53 stays in the cytoplasm when you treat the cells with the drug? you can check this by doing an immunofluorescence staining of p53 in the treated and non-treated cells



It implies that your transfection is not very good. 5 fold increase of endogenous protein may still be there, but the luciferase one is't being induced to the same level, therefore it is not working very well.

Note that luciferase experiments are fraught with difficulty and reproducibility issues.


Thanks dpo and bob1 for your time, interest and answer.
Here are more info 1)What I found that protein level was from nuclear protein (48 h treatment); 2) it may be because of very high basal level of p53-luc (Overnight/20 h treatment).

Is it because of lenght of exposure? Do I have to perform the luciferase for 48 h? why basal expression is very high?

Pls note that trasfection condition is well stablished one and easiest one to transfect HCT116 cells.

Thanks thanks
"Learning without thought is labor lost"

#5 bob1

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Posted 16 April 2009 - 04:46 PM

I presume you took the luciferase level after 48 hours of transfection, rather than adding the reagent and then 48 hours later measuring the activity - all the signal should have all gone within 10 minutes if you are using cell-titre glo system.

Measuring the activity 48 hours after transfection should be fine, it is a pretty standard time to do it and the activation of your transfected plasmid should be about maximal. p53 however, has a very short half-life (approx 30 min) so a peak after a treatment (e.g. drug, UV etc) could easily be missed. We have seen maximal p53 12 -14 hours after UV treatment of cells and a similar response to DNA damage drugs.

A high basal level of p53/luc implies that the protein is being constituitively expressed or that your cells are stressed all the time.

#6 Thapa

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Posted 17 April 2009 - 12:26 AM

I presume you took the luciferase level after 48 hours of transfection, rather than adding the reagent and then 48 hours later measuring the activity - all the signal should have all gone within 10 minutes if you are using cell-titre glo system.

Measuring the activity 48 hours after transfection should be fine, it is a pretty standard time to do it and the activation of your transfected plasmid should be about maximal. p53 however, has a very short half-life (approx 30 min) so a peak after a treatment (e.g. drug, UV etc) could easily be missed. We have seen maximal p53 12 -14 hours after UV treatment of cells and a similar response to DNA damage drugs.

A high basal level of p53/luc implies that the protein is being constituitively expressed or that your cells are stressed all the time.


bob1,
thank you so much for your nice reply. Also thank to share your own example.
In my case, I did transfection overnight (Lipofectamine+Plasmids in OptiMEM), recovary for 6 h in full medium and then next drug treatment in serum free condition for 24 h. I looked for induction in drug treatment group over vehicle (0.1% DMSO) treatment at same time and condition. I measured finally using Dual luciferase system (Promega)...thats all and sounds so simple.

So, what you think If I try to reduce time of trasfection 3-5 h followed by recovary overnight, reduce stress on cells (treating drug in full medium), and decrease drug exposure time to 12 h???

Warmest regards,
Thapa
"Learning without thought is labor lost"

#7 bob1

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Posted 17 April 2009 - 04:08 PM

0.1% DMSO is quite high, there will still be cytopathic effects at this level. Try diluting it further - make a more concentrated stock.

I would keep the transfection time the same. Drug treatment depends on the drug, some will be immediately effective, others not so fast. You need to do a time course experiment and look for maximal activation.

#8 Thapa

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Posted 18 April 2009 - 12:19 AM

0.1% DMSO is quite high, there will still be cytopathic effects at this level. Try diluting it further - make a more concentrated stock.

I would keep the transfection time the same. Drug treatment depends on the drug, some will be immediately effective, others not so fast. You need to do a time course experiment and look for maximal activation.


Yeah bob1,
its an another good idea of doing time course expt. Let me try according to your suggesion at least one time. Thanks for all your suggestions.

Best regards,
Thapa
"Learning without thought is labor lost"




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