Hi all,
I would like to ask you a question about the data analysis in an enzymatic activity assay. It is the first time I deal with this kind of stuff. I have a kit for the beta lactamase activity assay. I attached the leaflet of the kit. When it comes to make calculations to find the beta lactamase activity I can't understand very well a few steps ("Data analysis"and "calculations", pag 15 and 16). In particular, it says to calculate the time points A1 and A2 with the following formula(step 13.5)
𝑇𝑖𝑚𝑒 𝑝𝑜𝑖𝑛𝑡 𝑣𝑎𝑙𝑢𝑒 = (𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 ‒ (𝑦 ‒ 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡)/𝑆𝑙𝑜𝑝𝑒 )
I have a little bit confusion. Are they the absorbance values of the unknown concentration samples or of the standard? Why do I need to calculate them if I read these values directly at the spectrophotometer? The same for the step 13.7..it says to calculate the B nmoles with the formula
𝐵𝐿 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = ( 𝐵/(𝑇2 ‒ 𝑇1) × 𝑉)) ∗ 𝐷
Why do I need to calculate the B nmoles?Can't I get this concentration looking at which concentration the Absorbance of my unknown sample correspond on the standard curve?
Also, I can't find online a practical example of this procedure with numerical values and graphs that can help me to understand each single step. Please could you suggest me some document to look at?
Thank you very much
Hi there,
Time point value corresponds to nmoles detected at a given time of the measurement. It actually consists in a variation of absorbance divided by the slope of the standard curve (which is variation of OD per nmol of product).
B is the difference of time point values between T2 and T1 and the calculation gives the activity expressed as nmol/mn/mL.
Hi
Briefly, when you want to had standard curve you have to drawing a chart that contains the values of your samples, any amount that the range of this chart is closest to the sample values is the correct answer and in the other hand situation of your exam maybe be very different.
So in each exam you need different point in standard curve and your curve will be variable.
Thanks for your answers. What I don't fully understand is Are T1 and T2 the initial and the final time of the measures?and A1 and A2 are taken from the standard curve?
Yes and both are same.
first you use multiple T to drawing standard curve and then use for sample and match with standard curve.
T1 and T2 are 2 times of measurement of the kinetics between which absorbance varies linearly from A1 and A2. So it's all about kinetics experiment. Standard curve only serves when converting absorbance into nmol.
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays