luciferase assay - How to adjusting parameters - (Dec/24/2007 )
i have been doing luciferase assays nowadays
i use allready fixed 15 sec time period for taking readings and 100 micro litres of luciferase reagent but if i then and there take reading of same sample again just by pressing 'go' key again then my reading increases slightly (some times more)
please tell what parameters i should start changing.
we use time period 30 sec for taking readings.
I don't think you need to adjust the parameters for luciferase assay. the fact is after adding cell lysate to luciferase substrate, luciferase activity changes with time (decreasing). So it is very important to keep the time between adding lysate to the substrate and measuring the same across all samples.
The machine measures and adds the total counts together across the time window that you set. The reaction rate is not linear over time. It starts with an increased rate, peaked out, then drops over time.
I agree with BBname and I normally set the time to 10 sec, because I have a lot of samples. :-)).
hey thanku folks
but what about the ratio of cell lyasate and luciferase assay buffer
ever tried manipulating that ?
The standard protocol uses 10 to 20 ul lysate and 100 ul substrate mixture. I have used as much as 50 ul of lysate and as low as 50 ul of substrate in some systems for various reasons (low luc counts, cut the cost per assay, etc). You need to pre-test it first to see if that will work for your system.