luciferase assay (View forum version)



smilealie

Posted 22 December 2009 - 06:43 AM

hello every one,

i am not understanding about luciferase assay.. why we should normalization doing beta gal assay?
if any body have the information kindly let me know.it will be help full...
thank you
alie.

bob1

Posted 22 December 2009 - 03:44 PM

B-gal is used as normal cells don't have much B-gal activity (unless the population is senescent) - so plasmids containing B-gal can be transfected and used to compare between samples to show that a similar amount of the the cells were transfected.

You don't have to use B-gal, you could use something like chlorampehnicol acetyltransferase, or even do some real time PCR to look at transfection levels.

KevinK

Posted 29 December 2009 - 09:49 AM

Smilealie,
It is typical to normalize a luciferase reporter gene assay with a seperate reporter to help give an internal reference for all samples. As bob1 mentioned, variations in transfection efficiency, cell viability or other factors can lead to false interpretation of results. Also, the interanl control can help reveal non-specific events that influence a reporter, again helping to understand the specific influences on the experimental reporter assay. For instance, a titration of a compound may increase reporter expression at low concentrations, but be toxic at higher concentrations. Without the internal control, it would be difficult to see the trend in toxicity. I have co-authored an article on the topic, which can be found here: http://www.promega.c...7/cn017_09.pdf.

Please let me know if you have any additional questions, and you can always feel free to contact Promega Technical Services.

Regards,
Kevin

smilealie

Posted 05 January 2010 - 07:53 AM

thanks kevin.it was a nice information.if i have any question i wil contact you.

thank you,
alie

Smilealie,
It is typical to normalize a luciferase reporter gene assay with a seperate reporter to help give an internal reference for all samples. As bob1 mentioned, variations in transfection efficiency, cell viability or other factors can lead to false interpretation of results. Also, the interanl control can help reveal non-specific events that influence a reporter, again helping to understand the specific influences on the experimental reporter assay. For instance, a titration of a compound may increase reporter expression at low concentrations, but be toxic at higher concentrations. Without the internal control, it would be difficult to see the trend in toxicity. I have co-authored an article on the topic, which can be found here: http://www.promega.c...7/cn017_09.pdf.

Please let me know if you have any additional questions, and you can always feel free to contact Promega Technical Services.

Regards,
Kevin