I would like to quantify GUS activity in rice leaf. Below are the protocol, but I have questions in some of the steps:
1) Prepare protein extraction buffer (50mM Phosphate Buffer pH 7, 0.1% SDS, 0.01M EDTA pH 8, 0.1% Triton X-100 and B-mercaptoethanol 10mM) and store in 4C. Extract protein from the rice leaf
2) Prepare Stop Reagent 0.2M sodium carbonate.
- should I store this in 4C or room temperature?
3) Prepare 2mM MUG stock
- Can I prepare this using the extraction buffer that I use for protein extraction above?
- should I keep in 4C or -20C?
4) Prepare 1mM 4-MU stock for standard by adding 4-MU powder in dH2O. Take 10uL and add into 10ml water to obtain 1uM 4-MU stock. Keep both stock in 4C in dark. Dilute to 10, 25, 50, 100, 150, 200nM series using Na2CO3 for std.
- Can I prepare 1mM 4-MU and 1uM 4-MU in water or should I use sodium salt?
5) Dilute 2mM MUG stock to 10mM using protein extraction buffer. Prepare fresh everytime
6) Take extracted protein out from -80C and thaw on ice. Dilute 100uL extracted protein using 400uL protein extraction buffer (pre-warmed at 37C)
- should I keep this diluted protein at 37C as well?
6) Add the diluted protein to 500uL of 10mM MUG pre-warmed at 37C. Invert mix and incubate at 37C
7) Take 200uL from the mixture at 0, 15, 30, 35, 60 min and add into 800uL 0.2M Na2CO3, invert mix. Put at RT in dark.
8) Transfer to microplate
9) Excitement 365nm Emission 455nm, 10nm slit.
10) Result : nmol MU produced/reaction interval time/ug protein.
Thank you
In general, high concentration inorganic buffers can be stored at room temperature, but solutions of sensitive compounds are best stored either in the fridge, or aliquoted and stored deep-frozen. Avoid freeze-thaw cycles. In dilute solutions (less than, say, 100 mM) bugs can grow, these are best refrigerated. Bottles of substances that are sensitive to light are best wrapped in aluminum foil, this includes all fluorescent compounds.
Working enzyme solutions are made as needed for one day, and stored on ice. It is good practice, for example when measuring v as function of [S], to measure once with decreasing [S], and then a second time increasing. Plot both data sets on the same plot and look for systematic differences that may indicate enzyme inactivation over time.
Step 5 is definitely false, you can't prepare a 10 mM solution from 2 mM by dilution. Could that be 10 µM?
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques
- Analytical Chemistry
- pH