Activity determination of endo-PGase detailed protocol
Microplate assay for endo-polygalacturonase activity determination
based on ruthenium red method.
Ortiz et al.
Materials
96-wells thermal cycle device, 96-wells PCR plates, Adhesive sealing films for 96-wells PCR plates, Multichannel pipet, Repeating pipette, Microplate reader device with 540 nm filter, Rutenium Red (RR), Citrate phosphate buffer (CPB), Polygalacturonic acid (PGA), Sodium hydroxide.
Procedure
Use reverse pipetting in all steps to minimize the error and keep the samples all the time in cold water or ice rack.
Testing suitable sample dilutions
1. Building the curve by duplicate. In the file G and H of the PCR plate add 16 µL per well of each standard solution.
2. Make a six serial dilution of each sample (enzyme) in buffer pH 5 50 mM CPB.
3. Use the files A, B, C, D, E and F of the PCR plate to add 8 µL of each serial dilution.
4. Add 8 ul of 0.5 %% PGA in the tube wallIn each well containing the diluted sample to be tested (without touching the sample).
5. To mix the reagent with the samples centrifuge at 2500 rpm x 1’.
6. Place the plate in the thermal cycler and incubate using the following program (40 °C x 20' and 4 °C x 1').
7. Add to each well 40 µL of RR solution. (use a repeating pipette to step)
8. Add to each well 100 µL of 8 mM NaOH. (use a repeating pipette to step)
9. Cover the PCR plate with film, place the sealed plate on a support for mixed, you can use a rack or an old flat bottom (ELISA) plate.
10. Mix for 30'' using a vortex mixer.
11. Centrifug the plate at 2500 rpm x 10'.
12. Take 25 µL of the supernatant and transfer to a flat bottom plate (ELISA) preloaded with 175 µL of H20.
13. Read the absorbance at 540nm, activate mixed function for 30'' prior to reading.
The dilution whose absorbance is found in the middle of the standard curve range is suitable for testing.
Activity determination
1 Building the curve by duplicate. In the file G and H of the PCR palte add 16 µL per well of each standard solution.
2 Dilute conveniently each sample (enzyme) in buffer pH 5 50 mM CPB.
3 In the files A, B and C of the PCR plate add 8 µL of each diluted test sample.
4 To sample blanks repeat the step 3 using the files D, E and F of the PCR plate.
5 In each well containing the sample to be tested add on the tube wall (without touching the sample) 8 µL of 0.5% PGA.
6 To mix the reagent with the samples centrifuge at 2500 rpm x 1’.
7 Place the plate in the thermal cycler and incubated using the following program (40 °C x 20' and 4 °C x 1').
8 Add 40 µL of RR solution in the files A, B and C of the PCR plate to stop the reaction. (use a repeating pipette to step)
9 Add 8 µL of 0.5% PGA at the files that contain the sample blanks (D, E and F) and immediately add 40 µL of RR solution . (use a repeating pipette to step)
10 Add 8 µL of 0.5% PGA at the files that contain the curve (files G and H). (use a repeating pipette to step)
11 Add to each well 100 µL of 8 mM NaOH. (use a repeating pipette to step)
12 Cover the PCR plate with film, Place the sealed plate on a support for mixed, you can use a rack or an old flat bottom (ELISA) plate.
13 Mix for 30'' using a vortex mixer.
14 Centrifuged the plate at 2500 rpm x 10'.
15 Take 25 µL of the supernatant and transfer them to a flat bottom plate (ELISA) preloaded with 175 µL of H20.
16 Read the absorbance at 540nm, activate mixed function for 30'' prior to reading.
For calculations using the formula detailed in the manuscript
Solution of 0.5% PGA
Make a solution of 0.5% sodium polygalacturonic acid in 50mM citrate phosphate buffer pH: 5
Standard curve
PGA µg / µL of 0.5% PGA in 50mM BCP / µL of 50mM CPB pH: 540 / 500 / 500
36 / 450 / 550
32 / 400 / 600
27.7 / 350 / 650
24 / 300 / 700
20 / 250 / 750
16 / 200 / 800
12 / 150 / 850
8 / 100 / 900
5.3 / 75 / 925
2.7 / 50 / 950
0 / 0 / 1000
50 mM citrate phosphate buffer
X: 0.1 M citric acid solution and Y: 0.2 M Na2HPO4 solution
100 mL of CPBpH / X / Y
2.6 / 40.25 / 4.87
2.8 / 36.51 / 6.75
3 / 33.06 / 8.47
3.2 / 30.26 / 9.87
3.4 / 28.00 / 11.00
3.6 / 25.64 / 12.18
3.8 / 23.86 / 13.07
4 / 22.15 / 13.93
4.2 / 20.82 / 14.59
4.4 / 19.25 / 15.37
4.6 / 18.21 / 15.89
4.8 / 16.85 / 16.58
5 / 16.05 / 16.98
5.2 / 15.19 / 17.41
5.4 / 14.27 / 17.87
5.6 / 13.29 / 18.35
5.8 / 12.27 / 18.87
6 / 10.90 / 19.55
6.2 / 10.17 / 19.92
6.4 / 9.10 / 20.45
6.6 / 7.87 / 21.07
6.8 / 5.01 / 22.50
7 / 3.47 / 23.27