Leaf GUS Assay
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一、实验试剂
GUS Buffer (500 ml)
2.0478 g Na2HPO4
1.2688 g NaH2PO4 (=50 mM NaPi pH7.0)
10 ml 0.5 M EDTA (=10 mM)
0.5 g Triton X-100
0.5 g N-Lauroylsarcosine Sodium Salt (=0.1%)
Extraction Buffer (500ul per sample) Assay Buffer (100 ul per sample)
10 ml GUS Buffer 10 ml GUS Buffer
7 ul b-mercaptoethanol (=10mM) 7 ul b-mercaptoethanol (=10 mM)
4.08 mg 4MUG
Make Extraction Buffer, Assay Buffer fresh and keep on ice
4MUG = 4-methylumbelliferyl b-D-glucuronide
Also prepare:
Kontes homogenisers (for eppen tubes)
4 eppen tubes per sample
1:5 Bradford Protein Assay Reagent (200 ul per sample)
37℃ heat block
0.2 M Na2CO3
0.15 M NaCl
二、实验步骤
Sample Preparation
1. Harvest one small leaf (<1cm length) or two very small leaves per plant and place in an eppen
2. Add 150ul of Extraction Buffer and homogenise
3. Centrifuge for 10 min., 15 000 rpm, 4°C
4. Collect 100ul of the supernatant into a new eppen
5. Centrifuge for 10 min., 15 000 rpm, 4°C
6. Collect 50ul of the supernatant into a new eppen
7. Dilute the sample by adding 250ul of Extraction Buffer
8. Store the sample at 4°C (or -80°C for longer periods; loss of activity occurs at -20°C)
Protein Quantification
9. Prepare 10ul protein standards in the first column of the microtitre plate. Add 0, 0.5, 1.5, 2.5, 3.5, 4.5 and 5.5 ul of 1ug/ul BSA, respectively, to each well. Next add appropriate volumes of 0.15M NaCl to each well to bring the volume to 10 ul.
10. Add 200 ul of Bradford reagent to the standards. Also add 200ul of Bradford reagent to remaining wells that will contain your sample
11. Add 10 ul of each sample to each well (don't forget which well is which sample!)
12. Mix gently on the vortex mixer and leave at rt for about 15 minutes
13. Make sure there are no bubbles in the well and quantify using micro-plate reader. Standards and samples are same volume so concentrations can be determined directly from the standard curve (added sample volume = ug/10 ul)
14. If any samples are greater than the highest standard then those samples are too concentrated and need to be diluted in Extraction buffer. Further dilute your original sample and re-quantify.
GUS Assay
15. For each sample, add 90ul of Assay Buffer to an eppen tube and place in 37°C heat block
16. Incubation time is important, so start each reaction every 15 sec (or 30 sec if you prefer) by adding 10ul of sample to the respective eppen tube, quickly vortexing and returning to the heat block
17. Incubate samples for 30 min (60 min is also okay)
18. Stop each reaction by adding 900ul of 0.2 M Na2CO3, quick vortex and leave at rt (stop using same time increments used to start each reaction
19. Depending on your samples, you should also prepare one blank per sample (same as above but 0 min incubation)
20. Also prepare a tube for blanking the machine (10 ul Extraction Buffer, 90 ul Assay Buffer, 900 ul 0.2 M Na2CO3)
21. Measure fluorescence of the samples in the fluorescent spectrophotometer (number 19) and determine the nmol of 4MU in your samples. Divide this by the protein amount (same volume as quant so can take this directly) and the time to give assay as nmol 4MU/mg protein/min
