iGEM 2022 — Team csmu-taiwan


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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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# {{ self.title() }}

![](https://static.igem.wiki/teams/4151/wiki/paging/engineering-1.png)

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[Introduction](#introduction)
[AID gene modify](#C-terminus-NES-elimination-of-AID)
[Growth factor](#growth-factor)
[GFP toxicity](#gfp-toxicity)
[Concentration of doxycycline](#Concentration)
[References](#references)

# **Introduction**

---

Under the threat of the COVID-19 pandemic during our iGEM season, we seized every opportunity to work in labs and hoped to avoid mistakes to the utmost. However, our experiment process was still far from smooth because there were many problems, and we spent lots of time troubleshooting.

The first problem emerged when we designed our construct. We wanted to maximize the function of our target protein, AID. **AID works in many germinal center reactions (GCR), especially SHM and CSR**. Since our project focuses mainly on the production of monoclonal antibodies, we only required the **catalytic domain of SHM instead of CSR**. Thus, **we eliminated the 16 a.a of the C-terminus**, which includes the catalytic domain of CSR and the functional domain of NES. Removal of C-terminal a.a. can inhibit the function of CSR, but it might also affect the regulation and distribution of AID since **NES overlaps with the CSR catalytic domain**. We were concerned that the elimination of NES would change the diffusion patterns of the AID and even have adverse effects on the hybridoma cell. With the help of dry lab modeling, we can **predict the influence of NES deletion on AID nuclear diffusion and examine the effects on AID regulation**.

The second problem was that the hybridoma cell was not in good condition during culturing. Therefore, we made some adjustments to **modify the concentration of each ingredient** and test which provided the cell with the best living condition. With the help of dry lab modeling, we eventually found the best recipe for hybridoma to thrive.

After solving the cell culturing problem, we soon encountered another problem. The **cell survival rate was low after the lentivirus infection**. We searched for studies and designed several experiments to confirm that **the toxicity of the GFP reporter gene causes the cell to die**.

The final problem occurred when we were trying to figure out the best concentration of Tet-On system to express AID. To deal with this complicated problem, our dry lab designed a model to predict the relationship between doxycycline concentration and AID expression. Eventually, the result of our model corresponded to wet lab’s experimental outcome.

The **Design → Build → Test → Learn → Design process** in these problems was complicated but impressive, and we have detailed all the related information below. Please see the [Design](https://2022.igem.wiki/csmu-taiwan/design) page for more details about our thoughts.

![](https://static.igem.wiki/teams/4151/wiki/engineering/engineering.jpeg)

# **C-terminus elimination of AID**

---

## Design & build

In our construct design, we **eliminated the C-terminus 16 a.a, which can increase the efficiency of SHM while inhibiting the function of CSR** (see [Design](https://2022.igem.wiki/csmu-taiwan/design)). However, the **NES (nuclear export signal) overlapped the CSR**. Because the AID translated into the cytoplasm must diffuse into the nucleus to function, we were concerned that the elimination of NES would **change the diffusion patterns of the AID** or **even have a negative effect**. Therefore, we decided to **build a model for dNES AID**. We described **the kinetic process of nuclear transport of the AID** and **other transport factors with ordinary differential equations** to build a model. With this model, we **qualified the nuclear-cytoplasm exchange of the wild AID and the NES-deleted AID**. More details are described on the [Model](https://2022.igem.wiki/csmu-taiwan/model) page.

## Test

Before testing, we built a model describing the concentration of the expressed AID. Then, we incorporated this result into the NES model. After calculating by MATLAB, we got the following result (Fig. 1). The **blue line represents the diffusion of the wild AID**, while the **yellow line represents that of the NES-deleted AID**. In addition, the result also **shows the concentration change of nuclear transport factors**, such as importing, exportin-RanGTP complex, etc. More details are described on the [Model](https://2022.igem.wiki/csmu-taiwan/model) page.

![](https://static.igem.wiki/teams/4151/wiki/engineering/figure1.jpg)

**Figure 1.** The system of nuclear transport of original AID (blue) and NES-deleted AID (yellow).

## Learn

The **yellow line almost overlaps the blue line** (Fig 1.a), which means that **the deletion of the NES has no effect on the transport of AID** and **doesn’t change the concentration of AID in the nucleus** (Fig 1.b). The results(See [Model](https://2022.igem.wiki/csmu-taiwan/model)) gave us evidence that we could eliminate the C-terminus 16 a.a, **inhibiting the function of CSR without affecting the nuclear diffusion of the AID**. Therefore, we decided to continue using the construct of C-terminus eliminated AID.

# **Growth Factor**

---

## Design & build

We searched for all protocols to maintain the hybridoma offered by ERABIOTEQ ENTERPRISE CO., LTD, in good condition. After studying several researches, we found that **DMEM** and **RPMI** might be **two of the best culture medium candidate for our hydridoma cell**.

## Test

After testing RPMI-1640 and DMEM on our hybridoma, we discovered that the **hybridoma cell’s death rate in RPMI is lower than in DMEM**. However, the hybridoma cell did not **grow as fast as we expected in RPMI-1640**.

![](https://static.igem.wiki/teams/4151/wiki/engineering/figure-2-1.jpg)

**Figure 2-1.** Death rate of cell culturing in different medium for 48hr

| Medium | Cell mortality rate |
| --- | --- |
| RPMI | 14% |
| DMEM | 27% |

**Graph 2-2.** Cell mortality rate of each medium

## Learn

After consulting **professor, Mr. Jan, research studying**[1] and **modeling**(see [Model](https://2022.igem.wiki/csmu-taiwan/model)), we added some **growth factors** into our medium, such as **IL-6 and glucose**, to **trigger the growth of B lymphocytes**. During several rounds of discussion and testing, we eventually came up with our protocol of medium that provided **the hybridoma with the best living condition**. The concentration of each component is shown in the chart below.

![](https://static.igem.wiki/teams/4151/wiki/engineering/figure-2-3.jpg)

**Figure 2-3.**Growth rate of hybridoma under different glucose concentration in RPMI-1640.

![](https://static.igem.wiki/teams/4151/wiki/engineering/figure2-4.jpg)

**Figure 2-4.**Death rate of cell culturing in different medium for 48hr.

| Medium | Cell mortality rate |
| --- | --- |
| RPMI | 14% |
| DMEM | 27% |
| RPMI+IL-6 | 5% |
| DMEM+IL-6 | 23% |

**Graph 2-5.** Cell mortality rate of each medium

![](https://static.igem.wiki/teams/4151/wiki/engineering/figure-2-6.jpg)

**Figure 2-6.**Picture of the hybridoma recieved from ERABIOTEQ ENTERPRISE CO., LTD.

![](https://static.igem.wiki/teams/4151/wiki/engineering/figure-2-7.jpg)

**Figure 2-7.** Picture of the hybridoma culturing in RPMI-1640 + IL-6.

# **GFP toxicity**

---

## Design & build

To examine whether our target gene was successfully inserted into the genome of hybridoma cells and adequately expressed, GFP was chosen as our reporter gene. It can be observed easily under the fluorescence microscope.

## Test

Before the transfection of the lentivirus, which carries our target protein, we designed **lenti-GFP**, a lentivirus that carries only GFP, to **examine whether our protocol of lentivirus production and infection can function**. The fluorescence microscope result shows that the **lentivirus production** and **infection protocol works** since hybridoma expresses GFP adequately. However, we encountered **another technical problem**. The survival rate of the cell were low. To tackle this problem, we searched for possible causes and found **research[2] suggested that GFP might be toxic to immune cells** and **lead to the malfunction of specific cell lines or even death**. Since hybridoma originates from immune cells, we suggested that the **expression of GFP might result in a high cell mortality rate**.

## Learn

We discussed with IGEM NCHU and eventually determined that **RFP might be a better reporter gene for immune cells**. We constructed a **lenti-RFP** that carries only promoter and RFP gene to test whether it is helpful for cell viability. The graph below shows that using RFP as a reporter gene **increase the cell survival rate** and might be **a better reporter gene for immune cell**.

![](https://static.igem.wiki/teams/4151/wiki/engineering/figure-3-1.jpg)

**Figure 3-1.** Image of fluoresent microscope of hybridoma after infection of lentivirus carrying GFP.

![](https://static.igem.wiki/teams/4151/wiki/engineering/figure-3-2.jpeg)

**Figure 3-2.** Image of hybridoma under fluoresent microscope after infection of lentivirus carrying RFP.

# **Concentration of doxycycline**

---

## Design & Build

According to former research[3], the **higher the concentration of doxycycline**, the **faster the down stream sequences are expressed**. However, **high concentration of** doxycycline **may lead to cellular toxicity** and **interrupt with the proliferation of cell**.[4] To tackle with this issue, we performed **a modeling experiment to examine what is the best concentration for Tet-On system to fuction while did not disrupt the growth of cell**.

## Test

As shown in figure 6 of result from AID-expression model(See [Model](https://2022.igem.wiki/csmu-taiwan/model)), we demonstrated that **the amount of protein produced by Tet-On system under different concentration of** doxycycline(10X serial dilution). The result shows that **when the concentration of doxycycline exceed 10μg/ml, the amount of protein expressed is nearly identical**. According to this fiding, we **tested 0.1μg/ml, 1μg/ml, 10μg/ml of doxycycline in medium** and conducted western blot to **check the level of protein expression**.

![](https://static.igem.wiki/teams/4151/wiki/engineering/figure-4-1.jpg)

![](https://static.igem.wiki/teams/4151/wiki/engineering/figure-4-2.jpg)

**Figure 4.** The data of Western blot correspond with the Tet-On inducible model.

## Learn

As the model of doxycycline-inducible model shows, the **expression of protein under the concentration of doxycycline over 10μg/ml is nearly the same**. In comparison, the **result of the western blot shows that a medium with 10μg/ml of doxycycline has the best effectiveness of protein production**. According to the modeling data and the western blot result, we eventually decided to use **10μg/ml as the concentration of doxycycline to induce the Tet-On system**.

# **References**

---

1. Nakajima, K., Wall, R. (1990). IL-6 Induces Hybridoma Cell Growth Through a Novel Signalling Pathway. In: Potter, M., Melchers, F. (eds) Mechanisms in B-Cell Neoplasia 1990. Current Topics in Microbiology and Immunology, vol 166. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-75889-8\_8
2. Ansari AM, Ahmed AK, Matsangos AE, Lay F, Born LJ, Marti G, Harmon JW, Sun Z. Cellular GFP Toxicity and Immunogenicity: Potential Confounders in in Vivo Cell Tracking Experiments. Stem Cell Rev Rep. 2016 Oct;12(5):553-559. doi: 10.1007/s12015-016-9670-8. PMID: 27435468; PMCID: PMC5050239.
3. Das AT, Tenenbaum L, Berkhout B. Tet-On Systems For Doxycycline-inducible Gene Expression. Curr Gene Ther. 2016;16(3):156-67. doi: 10.2174/1566523216666160524144041. PMID: 27216914; PMCID: PMC5070417.
4. Ahler E, Sullivan WJ, Cass A, Braas D, York AG, Bensinger SJ, Graeber TG, Christofk HR. Doxycycline alters metabolism and proliferation of human cell lines. PLoS One. 2013 May 31;8(5):e64561. doi: 10.1371/journal.pone.0064561. PMID: 23741339; PMCID: PMC3669316.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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![](https://static.igem.wiki/teams/4151/wiki/paging/experiment.jpg)

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[Introduction](#introduction)
[Protocol](#protocol)

# **Introduction**

---

Here is the summary of all the experiments iGEM CSMU\_Taiwan 2022 designed and conducted this year. Overall, our experiments can be **divided into four parts**:

* Lentivirus infection (To bring our target protein, AID, into hybridoma cells).
* Western Blot (To confirm the expression of AID in hybridoma cells).
* ELISA (To test our antibodies' increased binding affinity).
* Sequencing (To identify mutation sites).

Although we only made it to the third session (ELISA) due to the **ongoing COVID pandemic in our country**, we were still optimistic about the **influence of our results** and would love to **share what we had done with all the iGEMers**! For more details, please refer to the bars below or see the [Proof of Concept](https://2022.igem.wiki/csmu-taiwan/proof-of-concept) page. As for the connection between our wet lab experiments and dry lab modeling, please see the [Design](https://2022.igem.wiki/csmu-taiwan/design) page and [Model](https://2022.igem.wiki/csmu-taiwan/model) page.

# **Protocol**

---

## Cell culture and aseptic operation

#### Materials

RPMI(with Glutamine & NaHCO3), FBS 10%, antibiotics (penicillin and streptomycin) 1%
Pipet, pipet aid, pipet tip, flask, cell needed (frozen in the liquid Nitrogen)

#### Methods

1. Turn on the UV light on the sterile operating table; turn it off before operation, then open the fume hood and wait for 1 minute.
2. Take out RPMI(with Gln & NaHCO3), FBS 10%, antibiotics (penicillin and streptomycin) 1%. Place FBS and antibiotics in a water bath of 37 degrees Celsius to preheat.
3. Sterilize the pipet, pipet aid, pipet tip, and flask. Put the equipment on the sterile operating table.
4. Mix RPMI, FBS, and antibiotics at a ratio of 100:10:1 to prepare the culture medium.
5. Take the culture tubes with the cells out of the liquid Nitrogen. Be sure to wear gloves when taking them.
6. After taking them out of the liquid Nitrogen bucket, loosen the cap of the culture tubes immediately to prevent them from breaking. Then put them in a water bath to heat them until nearly no ice is left in the tube.
7. Add 9ml of the culture medium, which has been prepared in step 4, to the flask. Then add 1ml of cells to it.
8. Put the flask into the incubator. When taking the flask out of the sterile space, be sure to lock it tight.
9. Slightly loosen the cap of the flask when putting it into the incubator so the gas generated by the cell can be discharged.
10. Take the flask out the next day to observe the state of the cells.
11. Change the culture medium every one to two days whenever it seems full. When changing the medium, remove 9ml of the cell-medium mixture and add 9ml of fresh, heated medium into the flask.

## Cell viability test

#### Materials

Cell suspension, trypan blue, a hemocytometer or a Coulter counter, a microscope, phosphate buffer saline (PBS), a centrifuge

#### Methods

1. Take 100 μl cell suspension and 100 μl trypan blue, and mix them well.
2. Take a small amount of the mixture (about 15 μl) and add it to the hemocytometer from the groove above the chamber, cover it with a cover glass, and observe it under a 100x inverted microscope. The live cells are not stained, while the dead cells are blue.
3. Count the total number of cells in the four squares, divide it by 4, then multiply it by the dilution factor. It should at least be multiplied by 2 because it is mixed with trypan blue in equal volume. Finally, multiply it by 10^4, then the number will be the number of cells in the cell suspension per ml. Therefore we can put it into a formula:

(Total cell number in the 4 large squares) x 2 x 10^4 / 4 = cells/ml

4. If not using the hemocytometer, we can also count the number of cells with the Coulter counter (Coulter Electronics), which can be used for automatic counting. However, it can’t distinguish between dead cells and living cells.
5. If there are too many cells in the sample, dilute it with phosphate buffer saline (PBS) before counting; if there are too many cell pellets that will interfere with staining and counting, centrifuge the sample at 1000 rpm, 5 min, room temperature to make counting easier.

## Lentivirus infection

#### Materials

Lentivirus, target cells, a 6-well plate, RPMI/IL-2(IL-2 10ng/ml), lenti-GFP(MOI=10), stock solution of the polybrene, an incubator, a centrifuge

#### Methods

1. Take the lentivirus out of the refrigerator and put it in a 37°C water bath to thaw it quickly.
2. Count the number of the cells at hand (see cell viability test section)
3. Take 10^5 cells and put them into a 6-well plate.
4. Add 900μl RPMI/IL-2(IL-2 10ng/ml) and 100μl lenti-GFP(MOI=10) into the plate.
5. Dilute the stock solution of the polybrene 1000 times to reach 8μg/ml, then add 1μl to the plate.
6. Put the plate into the incubator and let it sit for 10 minutes.
7. Seal the plate and centrifuge it at 2000rpm, 1hr, 25°C.
8. After centrifugation, leave it overnight in an incubator (37°C, 5% CO2).
9. On the next day, add an equal volume of RPMI/IL-2 (10ng/ml).
10. Continue to cultivate for about three days before harvesting the cells to observe.

## Sample preparation

#### Materials

A centrifuge, a small centrifuge, target cells, iced phosphate buffer saline (PBS), lysis buffer, protein inhibitor, microcentrifuge tubes (Eppendorf tubes), a pipet, a vortex

#### Methods

1. Turn on the centrifuge in advance to pre-cool.
2. Centrifuge the cells (1500rpm, 4°C) for 10 minutes.
3. Keep the supernatant for later use, such as testing for cell signals or antibodies.
4. Wash the centrifuged cells with iced PBS, and then centrifuge the solution again at 1500rpm, 4°C for 10 minutes.
5. Addprotein inhibitor in to lysis buffer (12mg/10ml) and vortex. After mixing, add 300μl / 5\*10^5 cells.
6. After the PBS is completely dry, add the lysis buffer mixture to the cell, then put it in a -80°C fridge for 5 minutes.
7. Take it out of the fridge. Transfer the solution into an Eppendorf tube, and centrifuge it at 13000rpm, 4°C for 30 minutes.
8. Use a pipet to suck the supernatant out and put it in another Eppendorf tube to isolate the protein.
9. Add the buffer sample and protein sample together in a ratio of 1:4.
10. Mix it well with a vortex, then centrifuge it with a small centrifuge for a few seconds.
11. Put it in a dry bath at 100°C for 8 minutes, then leave it on ice for later use.

## Western blot

#### Materials

The Western blot apparatus, three clean centrifuge tubes, ddH2O, glass plates(large and small), the gel casting stand, sponge, Vaseline, solution A, solution B, solution C, 10% SDS, 10% APS, a 15-well comb, explosion-proof clips, Eppendorf tubes, a small centrifuge, a vortex, a gel holder, a tank, fresh running buffer, reused running buffer, the marker, ice.

#### Methods

1. Preparation steps

1. Take the APS out of the -20°C fridge and warm it up in the water tank.
2. Prepare three clean centrifuge tubes, one for ddH2O, one for the running gel, and the other one for the stacking gel. All three tubes should be rinsed with tap water and then RO water.
3. Wipe the large and small glass plates clean with alcohol, then put the small plate on top of the large plate. Slide them into the green casting stand and shake them to ensure that the plates are even; then, clip them tight with the green clips on the casting stand. Apply Vaseline to the sponge at the bottom of the stand to prevent gel leaking.
4. Grab the ingredients below:

| Name | running gel(ml) | stecking gel(ml) |
| --- | --- | --- |
| ddH20 | 6.66 | 4.2 |
| Solution A | 5.33 | 0.9 |
| Solution B | 4 | X |
| Solution C | X | 1.687 |
| 10% SDS | 0.6 | 0.0675 |
| 10% APS | 0.08 | 0.0337 |

2. Making running gel

1. The ingredients are added to the centrifuge tube in the order from top to bottom in the table above. Whenever an ingredient is added, gently shake the tube to mix it well.
2. Pour the solution into the space between the glass plates. Mind the speed when pouring, and make sure there is no air bubble in the gel.
3. Stop pouring when reaching the bottom of the green section of the casting stand behind the glass plates. After pouring, add some ddH2O at a constant speed to press the surface of the gel flat.
4. Let it stand for an hour.

3. Making stacking gel

1. After the running gel has done polymerizing, dump out the ddH2O.
2. Start adding the materials of the stacking gel into a centrifuge tube one by one from top to bottom in the table above, then shake until it mixes evenly.
3. Quickly pour the solution on top of the solidified running gel until it reaches the top of the small glass plate; if there is any air bubble between the stacking gel, use a tip to remove it or push it to the edge. This step should be done quickly because the stacking gel solidifies fast.
4. Insert a 15-well comb into the stacking gel. When inserting, make sure that there is no bubble; otherwise, the well with bubbles can’t be used.
5. Let it stand for 15 minutes.

4. Running the gel

1. While waiting for the stacking gel to polymerize, take the sample and the lysis buffer-sample buffer mixture (see sample preparation step 9), and clip them with the explosion-proof clips. Put them into the 100°C dry bath for 5 minutes; press the cover tight when finished heating and take down the explosion-proof clips.
2. Let them cool for a minute, then mix the marker and the lysis buffer-sample buffer mixture at a ratio of 1:9. Spin the Eppendorf tube for a few seconds with a small centrifuge, then use a vortex to mix the solution well.
3. See if the stacking gel has solidified. If it does, place the gels inside the gel holder and have the short plate and wells facing the interior of the gel holder, then place the gel holder inside the tank. After the assembling is done, pour the fresh running buffer into the space between the two pieces of glass and wait for ten minutes to confirm that there will be no leakage. If it leaks, pour the running buffer into a clean container, and then pour it back in after adjustment.
4. After confirming that there is no leakage, pull out the comb smoothly. Try not to damage the well when taking the comb out.

5. Loading sample and running the gel

1. Load the samples into the well. First, load the marker into the third well; then transfer all the samples into the wells, 20μl for each well. Finally, load the marker again.
2. After loading all the samples and markers, add some ice into the tank, then pour the reused running buffer to nearly the same height as the top of the running gel.
3. Place the lid on top of the Western blot apparatus with the electrodes matching the corresponding color (black to black, red to red), then plug in the cables according to the corresponding color.
4. Run the gel for 30 minutes at 80V first, then switch to 100V and run for 90 minutes.
5. Start preparing the materials for transferring when the gel running is about to end. Put the PVDF membrane into a plastic box, add some methanol, and put it on the shaker to shake for 30 minutes to activate the transfer film.

## Transfer

#### Materials

An iron plate, RO water, alcohol, reused transfer buffer, the black fleece layer, the 'sandwich' cassette, the filter paper, the PVDF membrane (activated), the gel that has finished running, a Styrofoam box, ice, the transfer tank, milk powder, TBST, Eppendorf tubes, a plastic box, a vortex, a shaker, a plastic dropper

#### Methods

This part takes three days to complete.

1. The first day

1. As the gel running ends, start making the “transfer sandwiches”. Take an iron plate, rinse it with RO water, spray alcohol on it, and wipe it dry. Pour the reused transfer buffer into it, then soak the black fleece layer, the ‘sandwich’ cassette ( including a black side and a transparent side), and the filter paper into the transfer buffer.
2. Take the black side of the cassette, and stack these materials on top of it in the following order: a layer of black fleece, a layer of filter paper, the PVDF membrane, the gel that has finished running, a layer of filter paper, a layer of black fleece. Before transferring the gel from the gel holder to the ‘sandwich’, the stacking gel should first be removed. Also, remember to cut off a corner of the filter paper and the corner of the gel that is closest to the well that was first loaded, so we can orientate later. Make sure there is no bubble between the gel and the PVDF membrane, then press the transparent side of the cassette on top of everything and lock the cassette tight.
3. Prepare a Styrofoam box and some ice. Put the transfer tank inside the box, then insert the ‘sandwich’ that was finished in step B into the tank, with the black side of the cassette matching the black side of the tank. Next, Put some ice into the tank, then pour in the reused transfer buffer that was used in step A, and also some fresh transfer buffer. Last, let the gel transfer at 100V and 300mA for 140 minutes.
4. Start preparing the blocking buffer approximately 30 minutes before the transfer was complete. For each piece of PVDF membrane, weigh 0.5g of milk powder and put them in an Eppendorf tube; then, add TBST until the total volume reaches 10ml. Mix the solution well with a vortex, then shake it with the shaker in the fridge. The amount of solution in one tube is the amount of blocking buffer required for a piece of PVDF; see how many pieces to block to decide how much blocking buffer is needed.
5. After the transfer is finished, take a plastic box that is rinsed with RO water and then wiped dry. Pour in some TBST and carefully place the PVDF membrane into the box. Remember that the PVDF film’s front and back sides should be distinguished carefully after the transfer. Dip the film into the liquid with the front side facing down; the front side is the side that was in contact with the gel during the transfer.
6. Let it soak for a while, then dump the TBST. Take out the blocking buffer in the fridge and add it in with the PVDF membrane remaining face down in the box. Then put the entire box onto the shaker in the -4°C fridge and shake it overnight.

2. The second day

1. Rinse a plastic box with RO water. Pick up the PVDF membrane that was soaked in the blocking buffer yesterday with a clean tweezer, then put it face-down in the rinsed plastic box. Pour in TBST until the membrane was covered, and shake it at room temperature with a shaker three times, 10 minutes for each time.
2. Prepare the primary antibody solution. First, formulate 2.5% milk powder in TBST.Dump the TBST in the plastic box and add the primary antibody solution. As for the amount of the primary antibody that should be added, it is calculated according to the protocol provided by the manufacturer, which is usually very little, such as 1μl. Be aware that the antibody should be kept at -20°C, and cannot be placed somewhere hot otherwise the antibodies will be denatured.
3. Put the entire box on the shaker and shake at -4°C overnight.

3. The third day

1. Take a new 15ml centrifuge tube and a new plastic dropper. To recycle the primary antibody solution, use the dropper to suck up the solution and add it to the centrifuge tube. Mark the tube with the date and antibody name, and store it in the -20°C fridge. The recycled antibody can be reused once.
2. Next, pour TBST into the box until it covers the PVDF membrane, then put the box on the shaker to shake for 10 minutes at room temperature. Repeat this three times.
3. While waiting, prepare 0.25g of milk powder and add it to a centrifuge tube. Then add TBST to 10ml. Add this solution to the box when the shaking in step B is done, then add the secondary antibody. The secondary antibody should be added according to the ratio given by the manufacturer. After adding everything, put the box back onto the shaker and shake for another 2 hours.
4. When the 2 hours are over, repeat step B.
5. Prepare the materials for imaging. Turn on the gel-imaging machine in advance.

## Gel Imaging

#### Materials

solution A, solution B, ddH2O, a tweezer, alcohol, the PVDF membrane, the gel imaging machine

#### Methods

1. When doing gel imaging, coloring agents are needed. A piece of PVDF membrane needs 200μl of coloring agent, but a little more than needed is usually prepared. Once the reagent is formulated, the tube that contains it should be wrapped with tinfoil to avoid exposure to the light.
2. The formula of the coloring agents is as below, with the ratio of solution A, solution B, and ddH2O being 1: 1: 6.

| Name | dosage |
| --- | --- |
| solution A | 500μl |
| solution B | 500μl |
| ddH2O | 3000μl |

3. Prepare a tweezer, alcohol, the PVDF membrane, and the color reagent.
4. Start the machine and complete the steps below.

1. Turn on the main power, the computer, and press the two buttons (POWER and CCD) on the front of the machine.
2. Open the program on the computer that was linked to the machine.
3. Select the button “S1”, then at the ‘cooler’ section, select the one that says “cool up “. Wait until the ‘Temp’ shows about -7°C before putting the PVDF membrane into the machine.
4. For the first grid ‘exposure’, set the time interval at ‘Exp(s)’ based on the strength of the antibody and protein.
5. Select ‘browse’ and set up the place where the images should save, and be sure to select ‘autosave image’ so the file will be saved automatically.
6. The ‘Time Lapse’ in the middle can decide how many pictures would be taken, and the exposure of each picture will be superimposed.
7. After everything is set, put the PVDF membrane into the drawer of the machine, with the front side up. Suck up 200μl of the coloring agent with a pipet and spread it slowly and evenly across the film, then close the machine door and start gel imaging.

## ELISA

#### Materials

ELISA plate(96 well), the antigen, the coating buffer B, a shaker, TBST, BSA, Anti-Mouse IgG conjugate HRP in blocking buffer, sulfuric acid

#### Methods

1. Warm up the iced antigen and dilute it to the target concentration with Coating buffer B.
2. Add 100μL of the diluted antigen solution to each well of the ELISA plate, and put it on a shaker in a 4°C freezer to shake for a night.
3. Dump the solution in the well the next day. Add 100μL TBST to each well, and put it in a 4°C refrigerator to shake for 10 minutes; repeat this for 3 times. Next, formulate the blocking buffer (2.5%BSA in TBST) and add 200μL of it in each well. Then put the plate back onto the shaker in the 4°C freezer and shake it overnight.
4. On the next day, pour out the blocking buffer in the well, add 100μL TBST to each well, and put it in a 4°C refrigerator to shake for 10 minutes; repeat this for three times. Then, prepare the detect antibody (medium). Add this medium to the wells, 100μL for each well, and put the plate back onto the shaker at 4°C to shake for a night.
5. On the next day, pour out the blocking buffer in the well, add 100μL TBST to each well, and put it in a 4°C refrigerator to shake for 10 minutes; repeat this for three times. Then, prepare the Anti-Mouse IgG conjugate HRP in blocking buffer. The antibody is diluted in the ratio of 1:2000. Add this solution to the wells, 100μL for each well, and put the plate back onto the shaker at 4°C to shake for a night.
6. On the last day, dump the liquid in the wells, add 100μL TBST to each well, and put it in a 4°C refrigerator to shake for 10 minutes; this time, repeat this step for 5 times. After that, add 100μL of substrate to each well and wait for 15 minutes, then add the stop solution, which is sulfuric acid, and put the plate in the ELISA reader to read the value.

## Sequencing (Planned)

#### Materials

Hybridoma cells, RNA extraction reagent, reverse transcriptase, dNTPs, H2O, RNAse inhibitor, primers for PCR, PCR buffer, polymerase, TAE buffer, PCR Clean-Up and Gel Extraction kit

#### Methods

1. RNA extraction from hybridoma cells
2. Reverse transcription to cDNA
3. Nested PCR amplifications
4. Cloning of PCR products
5. Sequencing
6. Analysis of sequences

#### Reference

Meyer L, López T, Espinosa R, Arias CF, Vollmers C, DuBois RM (2019) A simplified workflow for monoclonal antibody sequencing. PLoS ONE 14(6): e0218717.
https://doi.org/10.1371/journal.pone.0218717

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