Team:NYMU-Taipei/Immobilization

Home

Immobilization

Cell Lysis1

We get the proteins we need by the following method:

  1. Liquid culture: Pick up a single colony in the plate with a tweezer holding a tip, then put the tip in the Erlenmeyer flask filled with 50ml of LB with chloramphenicol. Incubate at 37ºC overnight.
Fig 1. The liquid cultures after incubation (from left to right: eforCP, AmilCP, sfGFP, mCherry).
Fig 2. The liquid cultures under fluorescence imager (from left to right: eforCP, AmilCP, sfGFP, mCherry).
  1. Put the liquid culture in a 50ml centrifuge tube, then centrifugate for 20mins (8500 rpm, 4ºC ).
  2. Discard the supernatant.
Fig 3. The cell pellets after discarding the supernatant (from left to right: mCherry, eforCP, AmilCP, sfGFP).
  1. Resuspend the cell pellet with 5ml of TE.
  2. Add 1.25ml of 10mg/ml lysozyme.
  3. Refrigerate at -20ºC for at least one day.

Immobilization Methods

Binding with Fluorescent Proteins2,3

In order to popularize the examination to the public, we need to immobilize the protein on some low-cost materials which can be seen everywhere. After looking up the concerned information, we decide to use paper as the support material, and there are two ways which may immobilize the protein on the paper. One of them is glutaraldehyde crosslink, and the other is periodate oxidation.


We use some kinds of fluorescent protein to confirm that the immobilization method we found is feasible.


The goal at this phase is:

  1. To confirm that the processed papers do bind the protein better than the native ones.
  2. To know how long for the paper binding the protein is proper.
  3. To know how to conserve the product is appropriate.


➼ For Goal 1 & 2

For goal 1 and 2, the experiments are as below. We use sfGFP and eforCP as models.


Glutaraldehyde Crosslink

  1. Prepare three 2cm x 2cm chromatography paper, and we call them Alpha, Beta and Gamma.
  2. Alpha and Beta are processed as below, but Gamma skip this step.
    1. Add 100μl of 0.25mg/ml Chitosan on the papers, and have them dry at room temperature.
    2. Immerse the papers in the 2.5% glutaraldehyde for 2hr.
  3. Add 300μl of sterilized water on the three papers, then use a blotting paper to absorb the excess water by contacting the blotting paper with the bottom of the chromatography paper. Repeat this step again.
  4. Add the protein on the three papers as below:
    1. Alpha and Gamma: Add 100μl of the protein, and have it react with the papers for 30 mins.
    2. Beta: Add 100μl of the protein, and have it react with the papers for 60 mins.
Fig 4. The papers after adding eforCP (from left to right: Alpha, Beta, Gamma).
  1. Use the tweezers to hold the chromatography papers and put them in the petri dish which is filled with 8ml of sterilized water for about 1 sec. Then put it in the another petri dish which is filled with 8ml of PBST for about 1sec.
Fig 5. The Petri dish filled with H2O or PBST for washing the unbinding protein.
  1. Use the imager to observe the fluorescence on the paper.
  2. Measure the fluorescence intensity of the above-mentioned sterilized water and PBST.
  3. Put the three papers in the three 50ml centrifuge tubes which is filled with about 15ml of TE respectively, and put them in a 4ºC refrigerator.
  4. Take the papers out of the centrifuge tubes to observe the fluorescence under the imager periodically.

Periodate Oxidation

  1. Prepare three 2cm x 2cm chromatography paper, and we call them Alpha, Beta and Gamma.
  2. Add 300μl of 0.5M NaIO4 on Alpha and Beta, and put them in the dark for 30 mins. Gamma skips this step.
  3. Add 300μl of sterilized water on the papers, then use a blotting paper to absorb the excess water by contacting the blotting paper with the bottom of the chromatography paper. Repeat this step again.
  4. Add the protein on the three papers as below:
    1. Alpha and Gamma: Add 100μl of the protein, and have it react with the papers for 30 mins.
    2. Beta: Add 100μl of the protein, and have it react with the papers for 60 mins.
  5. Use the tweezers to hold the chromatography papers and put them in the petri dish which is filled with 7ml of sterilized water for about 1 sec.
  6. Add 300μl of 1.6mg/ml NaCNBH3 on Alpha and Beta, leave them for 15 mins.
  7. Use the tweezers to hold the chromatography papers and put them in the petri dish which is filled with 7ml of PBST for about 1sec.
  8. Use the imager to observe the fluorescence on the paper.
  9. Measure the fluorescence intensity of the above-mentioned sterilized water and PBST.
  10. Put the three papers in the three 50ml centrifuge tubes which is filled with about 15ml of TE respectively, and put them in a 4ºC refrigerator.
  11. Take the papers out of the centrifuge tubes to observe the fluorescence under the imager periodically.

➼ For Goal 3

For this part, we use sfGFP as a model.


Glutaraldehyde Crosslink

  1. Prepare four 2cm x 2cm chromatography paper, and we call them Alpha, Beta, Gamma and Delta.
  2. Add 100μl of 0.25mg/ml Chitosan on the papers, and have them dry at room temperature.
  3. Immerse the papers in the 2.5% glutaraldehyde for 2hr.
  4. Add 300μl of sterilized water on the papers, then use a blotting paper to absorb the excess water by contacting the blotting paper with the bottom of the chromatography paper. Repeat this step again.
  5. Add 100μl of sfGFP, and have it react with the papers for 60 mins.
  6. Use the tweezers to hold the chromatography papers and put them in the petri dish which is filled with 7ml of sterilized water for about 1 sec. Then put it in the another petri dish which is filled with 7ml of PBST for about 1sec.
  7. Use the imager to observe the fluorescence on the paper.
  8. Conserve the four papers as below:
    1. Alpha: Put in the 50ml centrifuge tubes which is filled with about 15ml of TE, and put it in a -20ºC refrigerator.
    2. Beta: Put in the 50ml centrifuge tubes which is filled with about 15ml of TE, and put it in a 4ºC refrigerator.
    3. Gamma: Put in the empty 50ml centrifuge tubes and put it in a -20ºC refrigerator.
    4. Delta: Put in the empty 50ml centrifuge tubes and put it in a 4ºC refrigerator.
Fig 6. The centrifuge tubes used to conserve the papers.
  1. Take the papers out of the centrifuge tubes to observe the fluorescence under the imager periodically. We put the papers on a glass plate and put them in the imager to observe the fluorescence.
Fig 7. The papers on the glass plate.
Fig 8. The imager we used to observe fluorescence.

Binding with mOR103-15

After using fluorescent protein to confirm (1) the immobilization method we chose does work and (2) the proper binding time, we proceed to do the most important part of the experiment. That is, immobilizing mOR103-15 on papers. The experimental procedure are as followings:

  1. Prepare eight 2cm x 2cm chromatography paper. For the experimental group, we call them Alpha1, Beta1, Gamma1 and Delta1. As for the control group, we call them Alpha2, Beta2, Gamma2 and Delta2.
  2. Add 100uL of 0.25mg/ml Chitosan on the papers, and have them dry at room temperature.
  3. Immerse the papers in the 2.5% glutaraldehyde for 2hr.
  4. Add 300μl of sterilized water on the papers, then use a blotting paper to absorb the excess water by contacting the blotting paper with the bottom of the chromatography paper. Repeat this step again.
  5. Add 100μl of mOR103-15 on the experimental group, and add 100μl of sfGFP on the control group. Then, we have the proteins react with the papers for 60 mins.
  6. Use the tweezers to hold the chromatography papers and put them in the petri dish which is filled with 7ml of sterilized water for about 1 sec.
  7. Add different concentration of heptanl on the papers:
    1. Alpha1 and Alpha2: one-thousand-times dilution
    2. Beta1 and Beta2: ten-thousand-times dilution
    3. Gamma1 and Gamma2: hundred-thousand-times dilution
    4. Delta1 and Delta2: one-million-times dilution
    All the dilutions are prepared by adding water to the different volumes of heptanal.
  8. fter adding heptanal on papers for about 5secs, we use the tweezers to hold the chromatography papers and put them in the petri dish which is filled with 7ml of sterilized water for about 1 sec.
  9. Use micropipette to transfer 1ml of the above water to 1.5ml centrifuge tubes, and use the mass spectrometer to measure the amount of heptanal.

Expectantly, the amount of heptanal in experimental group shoμld be less than that in control group.

Immobilization Results

Binding with Fluorescent Proteins

➼ For Goal 1 & 2

Glutaraldehyde Crosslink

(1) sfGFP4

We detect the fluorescence intensity (unit: RFU) of the sfGFP after we lyse the cell and that in the H2O and PBST which we used to wash off the unbinding protein to quantify the amount of the protein which is immobilized on the paper. The equation is: Binding percentage = (X-(Y-Z))/X * 100% (X: the fluorescence intensity after cell lysis; Y: the sum of the fluorescence intensity in H2O and PBST which used to wash the paper; Z: the sum of the fluorescence intensity in the H2O and PBST which was not used to wash off the unbinding protein.

Table 1. Fluorescence Intensity & Binding Percentage (485nm/510nm)
Name Original Blank Alpha Beta Gamma
Intensity (RFU) 2584 179.088 415.456 288.32 917.76
Binding Percentage (%) - - 90.85263 95.85263 71.41362
Note: Original refers to the fluorescence intensity after cell lysis. Blank refers to the sum of the fluorescence intensity in the H2O and PBST which was not used to wash off the unbinding protein. Alpha, Beta, Gamma refers to the sum of the fluorescence intensity in the H2O and PBST which used to wash the corresponding paper.

As the result shows, the binding percentage is Beta > Alpha > Gamma. This is quite reasonable since that Gamma is the unprocessed paper and that the protein on Beta have more time to react with the paper than that on Alpha.

Fig 9. Fluorescence observation with the use of the fluorescence imager (from left to right: Alpha, Beta, Gamma).

From Fig 9., we observe that just after the experiment was over, there are no significant different among the papers. However, Gamma lost most of its fluorescence on the next day. Though we can see that Alpha and Beta also lost some of its fluorescence, the amount is rather small compared to Gamma. The below is the line chart of the mean OD of the papers versus the dates. (measured by ImageJ and made by Excel)

Fig 10. The line chart of mean the OD.

From Fig 10., we can know that at first, the OD of the three papers are almost the same. However, the difference among them become bigger over time.


(2) eforCP5

We use eforCP to do this experiment for two times. Here are the result of the first time.

Table 2. Fluorescence Intensity (589nm/609nm)
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

The result is quite weird. The values are ether zero or about a thousand. In addition, the values of the blanks are unusually high. The second time experiment gives the similar result. We think that there might be some chemicals in H2O and PBST to affect the result substantially, and the effect of eforCP is hence covered.

Fig 11. Fluorescence observation with the use of the fluorescence imager
Fig 12. The line chart of the mean OD.

Here are the second time.

Fig 13. Fluorescence observation with the use of the fluorescence imager.
Fig 14. The line chart of the mean OD.

The results of eforCP looks like them of sfGFP. That is, Gamma lost most of its fluorescence on the next day. The difference between the two proteins is that Alpha and Beta lose eforCP more quickly than losing sfGFP.


Periodate Oxidation

Table 3. Fluorescence Intensity
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

The data is actually meaningless because of the below image.

Fig 15. The papers in the imager when the experiment is over (8/28).

It’s frustrating that there may be some chemicals in the process of periodate oxidation that would destruct the fluorescence of the protein. As we can see from the above image, Alpha and Beta have almost no fluorescence. Gamma (the paper which is unprocessed), on the other hand, has a little amount of fluorescence.

Our member who did this experiment also observe that the green pigment of sfGFP disappeared as soon as he add sfGFP on Alpha and Beta during the experiment, but the pigment maintain on Gamma until he put Gamma in PBST.

Though this method may not have negative effect on MOR103-15, the result of it with the use of sfGFP as a model contribute nothing to our project. Hence, we discard this method.


➼ For Goal 3

Fig 16. The papers in the imager when the experiment is over (9/5).
Fig 17. The line chart of the mean OD.

Though the mean OD measured by ImageJ shows no great difference among the four papers, we can notice that Gamma’s color became yellow-green ten days after the experiment, and Delta did so seven days after the color change of Gamma.

Binding with mOR103-15

Unfortunately, we failed to measre the amount of heptanal in water with the mass spectrometer in our school. The machine just could found the signal of heptanal. We first think the reason may be the too low concentration of heptanal. Hence, we dissolved heptanal in methanol to make a 10 times diluted solution and measured the solution with the mass spectrometer again. (Since heptanal is slightly soluble in water, it is impossible to make a 10 times dilution with water.) However, the machine still could not find the signal, either. We deduced that it might be the acidic environment in the machine that destructed heptanal and made the signal gone.

Conclusions

➼ Goal 1: to confirm that the processed papers do bind the protein better than the native ones

Because of the significant difference between Alpha and Gamma on the day of the experiment and the next day of the experiment, we are sure that our immobilization method does work.


➼ Goal 2: to know how long for the paper binding the protein is proper

Though the original immobilization method2 just leave the papers and the proteins reacting for 30mins, we found that 1hr is more proper for sfGFP and eforCP because the fluorescence on Beta can maintain longer than that on Alpha. Since we don’t know how long will it take for mOR103-15 binding with the chromatography paper, we supposed to have them reacting for 1hr to make sure that mOR103-15 is immobilized on the paper and will not be removed easily.


➼ Goal 3: to know how to conserve the product is appropriate

Though the original method2 didn’t say that they put the product in the buffer, our experiment results show that if we don’t put the papers in the buffer (here we use TE), the color of sfGFP will be yellow-green ten or so days after the experiment, which may indicate the denature of the protein.

As for the appropriate conserving temperature, we haven’t notice the significant difference between Alpha and Beta yet. This might imply that the temperature is not so important or that our observation time is not long enough.

References

  1. Maggie Bryans, L. R. Column Chromatography of Green Fluorescent Protein, <http://biomanufacturing.org/curriculum-resources/resources/standard-operating-procedures/column-chromatography-of-green-fluorescent-protein>
  2. Wang, S. et al. Paper-based chemiluminescence ELISA: lab-on-paper based on chitosan modified paper device and wax-screen-printing. Biosens Bioelectron 31, 212-218, doi:10.1016/j.bios.2011.10.019 (2012).
  3. Wang, S. et al. Simple and covalent fabrication of a paper device and its application in sensitive chemiluminescence immunoassay. Analyst 137, 3821-3827, doi:10.1039/c2an35266d (2012).
  4. Superfolder GFP, <https://www.fpbase.org/protein/superfolder-gfp/>
  5. eforCP, <https://www.fpbase.org/protein/eforcp/>