I'm a little bit confuse about the method to prepare 4-MU standard curve. Here's the method that i'm using (questions are in between the steps):
1) Prepare 1mM 4-MU. Dilute to 1uM 4-MU. Store at -20C. Dilute to 0, 10, 25, 50, 100, 150, 200nm for standard curve.
2) Add 100ul 4-MU (or extracted protein for sample) into 400ul of prewarmed protein extraction buffer.
3) Add 500ul prewarmed 2mM MUG.
4) Incubate at 37C. Take 200ul of the mixture and add into 800ul of Stop Reagent (0.2M Na2CO3) at 0, 15, 30, 45, 60 min. (For 0 min, add 100 ul 4-MU (or extracted protein) into 400ul protein extraction buffer, then add this into 800ul Na2CO3, then only add in 500ul MUG)
- I'm confused about this part. I've read some articles on GUS fluorometry but some of them didn't include the time interval, instead, just incubate for one specific time (eg: 1hr) then, measure.
- But if I use the 15min interval method, how do I calculate the GUS activity for standard curve and also for the sample? How do I plot the curve? Is the calculation below correct?
*For 4-MU standard curve (FI= fluorescent intensity):
For each concentration, plot a FI vs time graph, so since I have 7 dilutions for 4-MU standard, I will have 7 individual graphs.
From these graph, I calculate FI changes over time, so, with this data, I plot another graph for standard curve which is FI changes over time vs 4-MU concentration.
4-MU activity = [FI (60min) - FI (0min)] / [60min x 4-mu concentration ]
Is this calculation correct?
I'm not going to discuss details. I would like to pay your attention to a mistake, when the calibration curve is obtained by diluting a single stock solution. You will get in your calibration a so called "systematic error." You must prepare at least 4-5 stock solutions. If you use solutions prepared by dilution of a single stock solution, this would be a "one-point" calibration.
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