Team:NYMU-Taipei/Measurement

Home

Measurement

We do two kinds of measurement in our project. First one is measuring the fluorescence intensity in H2O and PBST which are used to wash the unbinding protein with Varioskan Flash spectral scanning multimode reader to know the binding percentage of our protein. Second one is measuring the mean OD on the binding papers with ImageJ to know the variation of proteins on the papers.

Measuring the Fluorescence Intensity

sfGFP (ex:485nm, em:510nm, unit: RFU)1

Glutaraldehyde Crosslink

Table 1. The Result of Glutaraldehyde Crosslink
Original Blank H2O Alpha H2O Beta H2O Gamma H2O Blank PBST Alpha PBST Beta PBST Gamma PBST
Replica 1 2584 4.706 3.955 2.275 8.977 1.323 8.236 1.807 20.49
Replica 2 - 3.851 4.134 7.039 10.51 1.313 8.246 3.675 17.82
Replica 3 - - 2.018 4.519 8.003 - 10.7 5.015 18.49
Replica 4 - - 8.34 5.422 10.59 - 9.66 4.875 19.17
Replica 5 - - 1.893 5.701 7.135 - 6.742 4.841 19.58
Average 2584 4.2785 4.093 4.83 9.46 1.138 8.89 4.18 19.22
Amplify - 684.56 654.88 772.8 1513.6 210.88 1422.4 688.8 3075.2
Table 2. The Result of Glutaraldehyde Crosslink
Name Original Blank Alpha Beta Gamma
Intensity (RFU) 2584 447.72 1038.64 720.8 2294.4
Binding Percentage (%) - - 77.131579 89.431889 28.534056

The data above the 7th row are the raw value. The followings are the explanation of each name used in this table:

  • Original: the supernatant without the cell pellet after cell.
  • Blank H2O: the blank value of the sterilized water.
  • Alpha H2O, Beta H2O, Gamma H2O: the sterilized water which is used to wash off the unbinding protein of the corresponding papers.
  • Blank PBST: the blank value of PBST.
  • Alpha PBST, Beta PBST, Gamma PBST: PBST which is used to wash off the unbinding protein of the corresponding papers.
  • Average: the average value of the data.
  • Amplify: equation is: x*32*2.5*2, the followings are the reasons:
    • *32: we immerse the papers in a petri dish which is filled with 8ml of sterilized water or PBST, and add 250μl of the luquid in a well of a 96-well plate to get the data above. Because fluorescence intensity is linearly proportional to concentration, we have the values times 8ml/250μl to get the undiluted values.
    • *2.5: we only use 100μl of “original” to get its fluorescence intensity; hence, we have the other values times 250μl/100μl to make their volumes be 100μl.
    • *2: we will add each H2O values with their corresponding PBST values, which means add two kinds of liquids together. However, we want the final volumes of the liquid is 100μl, so the original volumes of the two liquids must be 50μl. Thus, we have the values times 100μl/50μl to simulate this situation.
Fig 1. How the equation of “Amplify” means in a visualized way.
  • Intensity: the equation is: (H2Ovalue + PBSTvalue)/2, this is just like add same volumes of different concentration of fluid together.
Fig 2. How the equation of “Intensity” means in a visualized way.
  • Binding Percentage: the equation is: (Original – ((Alpha, Beta, or Gamma) - Blank))/Original *100

Periodate Oxidation

Table 3. The Result of Periodate Oxidation
Blank H2O Alpha H2O Beta H2O Gamma H2O Blank PBST Alpha PBST Beta PBST Gamma PBST
Replica 1 4.325 0.8377 5.107 8.166 5.186 3.51 2.134 10.44
Replica 2 3.075 3.977 13.74 4.746 1.838 2.411 1.986 8.922
Replica 3 - 1.652 6.768 10.56 - 3.296 1.901 10.77
Replica 4 - 3.435 13.92 13.35 - 2.6 2.957 10.91
Average 3.7 2.475425 9.88375 9.2055 3.512 2.95425 2.2445 10.2605

We didn’t process the data further since the data is actually meaningless because of the reason described on the Immobilization page of the experiment.

eforCP (ex:589nm, em:609nm, unit: RFU)2

Table 4. The result Of The First Experiment With Glutaraldehyde Crosslink Method
Blank H2O Alpha H2O Beta H2O Gamma H2O Blank PBST Alpha PBST Beta PBST Gamma PBST
Replica 1 1601 765.7 1597 733.1 1018 755.5 733.3 706.1
Replica 2 0 923.1 0 827.2 1193 0 0 1534
Replica 3 - 742.1 904.9 1040 - 924.7 699.7 757.7
Replica 4 - 890.6 0 837.8 - 0 1040 819.7
Replica 5 - 0 880 700.5 - 760.5 966.8 1267
Replica 6 - 0 0 816.5 - 0 0 770.3
Table 5. The result of the second experiment with glutaraldehyde crosslink method
Blank H2O Alpha H2O Beta H2O Gamma H2O Blank PBST Alpha PBST Beta PBST Gamma PBST
Replica 1 993 1044 1666 1270 1599 887.9 874 823.2
Replica 2 0 1096 2607 1005 810 0 0 0
Replica 3 0 1010 922 1311 1599 1098 1362 1571
Replica 4 0 855 0 1059 929.8 0 1420 1651
Replica 5 0 1149 1280 1784 1822 1090 1190 1188
Replica 6 0 822.6 0 979.8 940 0 0 1808

Here we do nothing more than exhibiting the raw data because of the weird result. However, the devices we used cannot be the reason because the result has no abnormality in other experiments. Thus, we tend to assume that there might be some chemicals in H2O and PBST which affect the results.

Measuring the Mean OD

The followings are the steps of how we use ImageJ to measure the mean OD:

  1. File → Open → choose the interested image
  2. Image → Type → 8-bit
  3. Edit → Invert
  4. Analyze → Calibrate → Function: Uncalibrated OD, and check the box of “Global calibration”
  5. Cut off the parts other than the single paper which is going to be measured
  6. Image → Adjust → Threshold → adjust the threshold to leave it cover the paper
  7. Analyze → Set Measurement → check the box of “Mean gray value” and “Limit to threshold” (Because of step 3 and 4, “Mean gray value” here actually means “Mean OD”)
  8. Analyze → Measure

The followings are the result of measurement:

sfGFP1

Table 6. The Raw Data of Glutaraldehyde Crosslink with sfGFP
Date Alpha Beta Gamma
2019/08/29 0.134 0.134 0.142
2019/08/30 0.064 0.087 0.039
2019/08/31 0.068 0.093 0.035
2019/09/01 0.073 0.094 0.042
2019/09/02 0.086 0.115 0.049
2019/09/03 0.084 0.111 0.048
2019/09/04 0.095 0.116 0.05
2019/09/05 0.083 0.11 0.046
2019/09/06 0.088 0.115 0.051
2019/09/07 0.08 0.102 0.047
2019/09/08 0.076 0.109 0.046
2019/09/15 0.086 0.116 0.05
2019/09/21 0.078 0.108 0.045
2019/09/29 0.094 0.119 0.061
2019/10/06 0.094 0.115 0.062
Note: For the meaning of Alpha, Beta and Gamma, please refer to the Immobilization page.
Fig 3. The line chart of glutaraldehyde crosslink with sfGFP.

One may be confused by the fluctuation of the mean OD. It is because though we do our best to make every variable in each image same, there are always something we cannot control, which contributing to the result that some images are generally brighter than others. However, we still can find out the downward trend of the mean OD, which means that the proteins on the papers become darker and darker or that some proteins are no longer immobilized on the papers.

Some people may be curious about the variation of our measuring period. The reason is that there are no big difference between one day and the next day; we thus think that we don’t need to measure it so frequently.

Table 9. The raw data of the experiment for investigating the appropriate reservation condition
Date Alpha Beta Gamma Delta
2019/09/05 0.15 0.164 0.157 0.17
2019/09/06 0.155 0.163 0.155 0.169
2019/09/07 0.152 0.156 0.139 0.167
2019/09/08 0.147 0.154 0.144 0.169
2019/09/15 0.147 0.158 0.135 0.173
2019/09/21 0.156 0.16 0.146 0.176
2019/09/29 0.145 0.157 0.134 0.165
2019/10/06 0.153 0.159 0.144 0.179
Fig 4. The line char of the experiment for investigating the appropriate storage conditions.

The result is reasonable because in this experiment, we leave the four papers react with sfGFP for 1hr. This period of time is long enough for proteins to be immobilized on papers, which is the conclusion of our previous experiment. However, the fact that there are no downward trend doesn’t mean that the four storage conditions are as appropriate as each other. Actually, there are color changes on Gamma and Delta, which cannot be found out by measuring the mean OD. For the original images, please refer to our Immobilization page.

eforCP2

First Experiment

Table 7. The raw data of the first experiment with eforCP.
Date Alpha Beta Gamma
2019/08/30 0.101 0.11 0.092
2019/08/31 0.065 0.076 0.023
2019/09/01 0.063 0.074 0.024
2019/09/02 0.069 0.075 0.032
2019/09/03 0.064 0.077 0.035
2019/09/04 0.06 0.073 0.034
2019/09/05 0.056 0.071 0.033
2019/09/06 0.059 0.067 0.032
2019/09/07 0.059 0.068 0.032
2019/09/08 0.055 0.064 0.03
2019/09/15 0.053 0.063 0.033
2019/09/21 0.052 0.062 0.03
2019/09/29 0.057 0.065 0.036
2019/10/06 0.062 0.069 0.043
Fig 5. The line char of the experiment for investigating the appropriate storage conditions.

Second Experiment

Table 8. The raw data of the second experiment with eforCP.
Date Alpha Beta Gamma
2019/09/04 0.089 0.1 0.094
2019/09/05 0.053 0.073 0.031
2019/09/06 0.049 0.067 0.03
2019/09/07 0.045 0.063 0.029
2019/09/08 0.04 0.06 0.029
2019/09/15 0.044 0.047 0.031
2019/09/21 0.032 0.045 0.025
2019/09/29 0.04 0.046 0.03
2019/10/06 0.051 0.05 0.039
Fig 6. The line char of the experiment for investigating the appropriate storage conditions.

The main difference among the results of sfGFP and eforCP is that the downward trends are more obvious in the experiments which were did with eforCP, which may means that eforCP become darker quicker than sfGFP or that papers have more tendency to lose eforCP than sfGFP, so the decaying of mean OD of eforCP can hide the variation of uncontrollable variables.

References

  1. Superfolder GFP, <https://www.fpbase.org/protein/superfolder-gfp/>
  2. eforCP, <https://www.fpbase.org/protein/eforcp/>