I'm doing BSA test and I need a blank to identify the product of my substrate (which means I can't use water since BSA will read my enzyme as protein as well) ; problem is, once I substract the sample result with the blank, the result is totally in mess.
My sample consists of substrate, buffer, and enzyme, while the blank is buffer and enzyme only. Both are treated exactly the same + with the same parameter (pH, temp, and time). Is there anything wrong with this procedure / how was it supposed to be done?
Thank you
Hi,
I think you should have the followings 1. blank(buffer) 2. substrate control(buffer+substrate) 3.Enzyme control(enzyme +buffer) 4.The reaction (enzyme+substrate+ buffer). Then, subject them to the same condition using same buffer of optimum pH and temperature.Measure the absorbance.
I hope this will help you.
All the best!
@adesolajuliustola Thank u so much for reply! :)
when I have four, which one should I substract the number 4 from?
The number 2 and 3 is very big, if I substract 4-(1+2+3) it definitely will be minus
Hi,
Firstly, you need to get enzyme only(5)=3-1(Enzyme control-blank), then calculate the kinetic (4-5-2)(The reaction-enzyme only-substrate control).You can set your value to zero when t=0, (if you have a -ve value like -23 then you add +23 to get 0 when t=0).Furthermore, plot your graph Absorbance against time in order to get your slope(Vo) after adjusting your curve to have linear graph.
I hope this will help you.
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