1. Harvest tissue (can be stored at -70C)
2. Grind tissue in 100ul extraction buffer.
3. Spin 1min. Take supernatant, put on ice.
4A. 20ul for BCA assay (add 500ul assay reagent—-> 37C 30min
4B. Add 0.05ul mercaptoethanol to the remnant 80ul, spin 10min.
5. Set up GUS assay:
In borosilicate tube (VWR 60825-550) combine:
Plant extract: 20ul
Assay mix: 80ul
Mix well. Assay mix is 70 ul extraction buffer and 10ul substrate
6. Incubate at 37C covered with parafilm for 1 hour exactly.
1.Stop FUS assay by adding 900ul 0.2M sodium carbonate solution.
2.Measure fluorescence in Sequoia-Turner fluorimeter using a MU standard
as a control.
Fluorimeter must warm up for 15 minutes before use.
Gain 1 or Gain 10 should be sufficient.
Substrate solution: 1mM MUG in extraction buffer (22mg per 50ml)
Dissolve MUG in DMF first before adding to extraction buffer.
Store at 4C, but it is not stable for more than a few weeks.
50mM sodium phosphate pH7.0
10mM beta-mecaptoethanol (can also try 1mM fresh DTT)
2.12g sodium carbonate in 100ml water.
Mix reagents A and B as indicated 50:1 just before use. Make a standard
of 0, 5, 10, 20ug BSA (in 1ml). Use 5, 10, 20ul protein solution per
assay and add 500ul assay reagent. Incubate for 30min at 37C, then read
Absorbance at 562nm.
MU is not stable in solution and has to be prepared fresh, dissolved in
DMF and diluted to 1mM in extraction buffer, every time the standard is
9nmole MU in 1ml stop buffer will give a reading of 1559 at gain 1.
X=8.56 X 10(exp-3) X f/A
X=GUS activity in [pmole/min/ug protein]
A=absorbance of BCA assay assuming that 20ul protein was assayed.