LeafGUSAssay

實驗概要

a protocol for Leaf GUS Assay 

• This protocol is for small samples (usually single leaf from 21DAI plants), scale up for larger samples

• As there are usually many samples, we use a microtitre plate and plate reader for quantifying the protein samples

主要試劑

GUS Buffer (500ml)

    2.0478g   Na₂HPO₄

    1.2688g   NaH₂PO₄ (=50mM NaPi pH 7.0)

    10ml    0.5M EDTA (=10mM)

    0.5g    Triton X-100

    0.5g     N-Lauroylsarcosine Sodium Salt (=0.1%)

Extraction Buffer (500ul per sample)                          Assay Buffer (100ul per sample)

   10ml            GUS Buffer                                       10ml      GUS Buffer

   7ul        b-mercaptoethanol (=10mM)                        7ul        b-mercaptoethanol (=10mM)

                                                        4.08mg    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 (200ul per sample)

37℃ heat block

0.2M Na₂CO₃

      0.15M NaCl

實驗步驟

Sample Preparation

1.    Harvest one small leaf (<25px 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 5.5ul of 1ug/ul BSA,  respectively, to each well.  Next add appropriate volumes of 0.15M NaCl  to each well to bring the volume to 10ul.

10.Add  200ul of Bradford reagent to the standards.  Also add 200ul of Bradford  reagent to remaining wells that will contain your sample

11.Add 10ul 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/10ul)

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 0.2M 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 (10ul Extraction Buffer, 90ul Assay Buffer, 900ul 0.2M 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