Introduction

The overall experimental process of our project is as shown in the figure below. On this page, we will provide detailed explanations for each of the steps.

1. H-Nuclear Magnetic Resonance (NMR) spectroscopy

H-Nuclear Magnetic Resonance Spectroscopy (NMR) is an analysis method that uses the spin properties and magnetic moments of atomic nuclei. This method is mainly used to analyze the properties of materials.

The basic principles of H-NMR are as follows:

Certain atomic nuclei can have spin properties and magnetic moments. When these nuclei are exposed to a magnetic field, the energy levels of the rotating nuclei are separated. When energy levels are separated, the direction of the rotation axis changes as it transitions from a low energy level to a high energy level. At this time, the chemical surroundings affect the radio frequency range, and through this, it is possible to determine which atoms exist around the atom.

Fig. 3. H-NMR spectroscopy analysis conducted by Seunghun Kim at Yonsei University Center of Research Facility(YCRF)

Fig. 4. H-NMR spectroscopy analysis conducted by Seunghun Kim at Yonsei University Center of Research Facility(YCRF)

When peaks related to the structure emerge through NMR spectroscopy, the integral value of each peak is obtained, and the integral value is compared with the H-NMR table [1] to analyze which peak it is for, and through this, the structure can be analyzed.

Fig. 5. H-NMR spectroscopy peak data table

2. Protein-Aptamer Docking (PAD) experiment

To perform the PAD experiment, we first performed H-Nuclear Magnetic Resonance(NMR) spectroscopy to analyze the the structure of CEA protein and N56 aptamer. Furthermore, the structure of the protein-aptamer complex was also analyzed by H-NMR spectroscopy, the data was compared with the data before binding. That is, the H-NMR data for each protein aptamer before binding and the data for the binding complex were compared and analyzed. As a result, it was possible to analyze the binding pattern of the CEA and aptamer binding complex. Finally, the results of this analysis were visualized through a PAD experiment.

The imaging results analyzing the binding pattern of CEA protein and N56 aptamer are as follows.

Data deleted after the judging session

As we observed in PAD images, the bottom end of the aptamer is free to bind to the LFA sheet, and the N56 aptamer binds perpendicularly to the CEA.

To use aptamers in LFA, they must be bound nearly vertically, as shown in the image here.

Why is it important for an aptamer to exhibit nearly perpendicular binding to a biomarker to be considered suitable for LFA?

1.In any type of LFA sensor, the aptamer is fixed at one end. Chemically, the only location where the aptamer can be anchored arbitrarily while maintaining its function is at the end of the aptamer.

2.Various chemical repulsion forces, especially steric hindrance, occur very strongly near parts that have already been chemically bonded, that is, fixed. This is a very self-evident fact, and in one of the many papers reporting this, a Paper published in ACS Journal stated: "Due to steric hindrance, aptamers immobilized close to the surface may not be able to fold into the three-dimensional structure necessary for target recognition.” [2]

3.Therefore, It is very unlikely that a relatively large biomarker will be attached to the side of the aptamer immobilized on the LFA sheet by overcoming the enormous steric hindrance. (Of course, if the aptamer is extremely long and straight, the biomarker can bind to the side, but such aptamer is structurally very unstable and is not suitable for use as a probe.)

4.In other words, an aptamer suitable for LFA must bind a biomarker to one loop or end excluding the fixed end. When this is satisfied, this document expresses this as “binding almost vertically (or perpendicularly).”

In conclusion, the nearly vertical binding of the CEA protein and N56 aptamer was visualized through experiments. This experimental discovery demonstrates for the first time the potential application of an LFA assay-based CEA protein sensor using N56 aptamer, and these images are being released for the first time in iGEM.

3. Cell-Free Expression of CEA N domain

Cell-Free Expression

Cell-free protein synthesis was carried out using previously designed template DNA to minimize contamination that may occur in biological experiments and to quickly obtain proteins without using cells. A cell-free protein synthesis experiment was conducted using the ‘NEBExpress cell-free protein synthesis kit’, and 10 reactions were performed in the first step. Through this experiment, it was confirmed that the CEA N domain protein was expressed, but a sufficient amount of protein was not obtained to be used in the EMSA experiment. To secure additional CEA N domain protein, E. coli was transformed twice to obtain a DNA template. Afterward, a second cell-free protein synthesis was performed using the additionally obtained DNA template.

The preparations, protocol[3], and results of the experiment are as follows.

Preparations (Cell-Free Protein Synthesis)

NEBExpress S30 Synthesis Extract

Protein Synthesis Buffer (2X)
      
T7 RNA Polymerase (Supplied in 50 mM Tris-HCl, 100 mM NaCl, 20 mM β-ME, 1 mM EDTA, 50% glycerol, 0.1% Triton****™**** X-100, pH 7.9)

RNase Inhibitor, Murine (Supplied in 20 mM HEPES-KOH, 50 mM KCl, 8 mM DTT, 50% glycerol, pH 7.6)

NEBExpress Control DHFR-His Plasmid (Encoding E.coli dihydrofolate reductase)

Incubator

Pipette

Microcentrifuge tube

Cell-Free Expression protocol

1.Thaw all components (NEBExpress S30 Synthesis Extract, protein synthesis buffer (2X), T7 RNA polymerase, RNase inhibitor, Murine, and NEBExpress control DHFR-His plasmid) on ice.

2.Gently vortex the NEBExpress S30 Synthesis Extract and protein synthesis buffer to mix.

3.Combine reagents in a 1.5ml microcentrifuge tube on ice as follows:

      
        
          
            Components
            Negative control
            Positive control
            Sample
          
        
        
          
            NEBExpress S30 Synthesis Extract
            12 μl
            12 μl
            12 μl
          
          
            Protein synthesis buffer (2X)
            25 μl
            25 μl
            25 μl
          
          
            T7 RNA polymerase
            1 μl
            1 μl
            1 μl
          
          
            RNase inhibitor, Murine
            1 μl
            1 μl
            1 μl
          
          
            NEBExpress control DHFR-His plasmid (125 ng/μl)
            -
            2 μl
            -
          
          
            Plasmid template (>100 ng/μl)
            -
            -
            250 ng
          
          
            Water
            11 μl
            9 μl
            to 50 μl
          
        
      
4.Incubate reactions at 37℃, with vigorous shaking, for 2-4 hours.
5.Analyze by method of choice or freeze at -20℃ for later use.

  file[\kuas-seoul\updatefiles\NEBExpress_CFE_manual.pdf]

Components	Negative control	Positive control	Sample
NEBExpress S30 Synthesis Extract	12 μl	12 μl	12 μl
Protein synthesis buffer (2X)	25 μl	25 μl	25 μl
T7 RNA polymerase	1 μl	1 μl	1 μl
RNase inhibitor, Murine	1 μl	1 μl	1 μl
NEBExpress control DHFR-His plasmid (125 ng/μl)	-	2 μl	-
Plasmid template (>100 ng/μl)	-	-	250 ng
Water	11 μl	9 μl	to 50 μl

Preparations (Transformation)

DH10B (Plasmid cloning strain)

DNA template

Lysogeny broth (LB)

Shaker

Centrifuge 

LB plate

Glass beads

Transformation protocol

[Plasmid Cloning]

1. Add the 80 ng/µL of GOI (gene of interest) in the aliquoted competent cell.
2. Thaw on ice for 20 minutes.
3. Apply heat shock at 42˚C for 45 seconds.
4. Thaw on ice for 2 minutes.
5. Add 800 µL of warm LB on each tube.
6. Vigorously shake samples at 200 rpm, 37˚C at the shaker for 1 hour.
7. Centrifuge the tubes at 13,000 rpm for 1 min at RT.
8. Re-suspend 100 µL of warm LB.
9. Apply 100 µL of the solution on each LB plate (including antibiotics) with glass beads/spreader.

Result

We were able to successfully transform the CEA N domain gene sequence using the DH10B strain.

SDS-PAGE

SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is a technique for classifying proteins by molecular weight. SDS-PAGE eliminates the effects of protein structure and charge and simply classifies proteins based on molecular size. SDS is an anionic detergent that denatures proteins and destroys their structures. After SDS treatment, the protein is uniformly coated with a negative charge proportional to its molecular weight, and this coating neutralizes the intrinsic charge of the protein, making it simple to classify by size during electrophoresis. Polyacrylamide gels act as a medium by which proteins travel when exposed to an electric field.

SDS-PAGE was performed to check whether the protein was properly expressed after expressing the CEA N domain with the cell-free expression system. According to the reference paper[4], purified CEA N domain represents about 13 kDa band. In the case of KUAS' study, template DNA was designed to express 6x His tag together in the CEA N domain, so it could be expected that a protein band of approximately 16 kDa would appear.

The preparations, protocol[5], and results of the experiment are as follows.

Preparations (SDS-PAGE)


    
 PageRuler™ Unstained Low Range Protein Ladder 
            
 
            
 2 M Tris-HCl (pH 8.8)
            
 1 M Tris-HCl (pH 6.8)
            
 10% SDS (w/v)
            
 50% glycerol (v/v)
            
 1% bromophenol blue (w/v)
            
 
            
 1. Solution A (Acrylamide stock solution)
                
  30% (w/v) acrylamide
                
  0.8% (w/v) bis-acrylamide
                
 2. Solution B (4x separating gel buffer)
                
  2 M Tris-HCl (pH 8.8)
                
 10% SDS
                
 H₂O
                
 3. Solution C (4x stacking gel buffer)
                
  1 M Tris-HCl (pH 6.8)
                
  10% SDS
                
  H₂O
                
 4. 10% ammonium persulfate
 5. Electrophoresis buffer
                
  Tris
                
  Glycine
                
  SDS
                
  H₂O
                
6. 5x sample buffer
                
  1 M Tris-HCl (pH 6.8)
                
  50% glycerol              
  
  10% SDS
  
  2-mercaptoethanol
              
  1% bromophenol blue
                
  H₂O
                
            
 
            
  Solution A
            
  Solution B
            
  Solution C
            
  H₂O
            
  10% ammonium persulfate
            
  TEMED

SDS-PAGE protocol

 
 1. Combine NEBExpress Cell-free Protein Synthesis System reaction with 5x sample buffer. Also, prepare a negative control sample.
 2. Incubate at 100℃ for 5 minutes.
 3. Load 5 μl of the standard protein marker into the first lane.
 4. After a quick microcentrifuge spin, load 5 μl of samples directly onto the 15% gel. To ensure uniform mobility, load an equal volume of SDS-PAGE Blue loading buffer into any unused wells.
 5. Run the gel at 80-120V until the protein is completely separated by molecular weight.
 6. Stain with Coomassie Blue stain buffer overnight.
 7. Destain with destain buffer for overnight.
 8. Analyze the location and thickness of the band appearing in the gel.

Result

In the left gel, the SDS-PAGE results showed, in order, marker ladder, positive control, negative control, samples 2, 3, 4, 5, and 6. In the right gel, the SDS-PAGE results showed, in order, marker ladder, negative control, positive control, samples 1, 2, 3, 4, 5, and 6.

It was confirmed that the CEA N domain protein was not well expressed in the cell-free expression experiment.

In the upper gel, the SDS-PAGE results showed, in order, marker ladder, negative control, and positive control, samples 6, 7, 8, 9, 10, and 11. In the lower gel, the SDS-PAGE results showed, in order, marker ladder, negative control, positive control, samples 1, 2, 3, 4, and 5.

Through this result, it was confirmed that the CEA N domain protein was well expressed in the cell-free expression experiment, but not in sufficient quantities for use in the EMSA experiment.

Protein purification

The CEA N domain protein was purified for EMSA with N56 DNA aptamer. For rapid screening and purification of 6x His-tagged proteins from small-scale expression cultures, the protein was purified using a Ni-NTA spin column.

The preparations, protocol[6], and results of the experiment are as follows.

Preparations (Protein purification)

        (pH 8.0)

        50mM NaH2PO4
        
        300mM NaCl
        
        10mM imidazole
        
         (pH 8.0)
        
        50mM NaH2PO4
        
        300mM NaCl
        
        20mM imidazole
        
         (pH 8.0)
        
        50mM NaH2PO4
        
        300mM NaCl
        
        500mM imidazole
        
        Benzonase endonuclease 25 U/µL
        
        Lysozyme stock solution 10 mg/mL in water
      
        Ni-NTA spin columns

Protein purification protocol

1. Incubate columns for protein purification on ice for 15-30 minutes
2. Centrifuge protein sample at 12,000 x g for 15-30 min at 4℃. Collect supernatant.
3. Equilibrate the Ni-NTA spin column with 600 µL buffer NPI-10. Centrifuge for 2 min at 890 x g (approx. 2900 rpm).
4. Load up to 600 µL of the protein sample containing the 6x His-tagged protein onto the pre-equilibrated Ni-NTA spin column. Centrifuge for 5 min at 270 x g (approx. 1600 rpm), and collect the flow-through.
5. Wash the Ni-NTA spin column twice with 600 µL buffer NPI-20. Centrifuge for 2 min at 890 x g (approx. 2900 rpm).
6. Elute the protein twice with 300 µL buffer NPI-500. Centrifuge for 2 min at 890 x g (approx. 2900 rpm), and collect the eluate.
7. To confirm whether the protein is well purified, make a protein sample using part of the purified protein, perform SDS-PAGE, and check the results.

  file[\kuas-seoul\updatefiles\EN-Ni-NTA-Spin-Kit-Handbook.pdf]

Result

The protein produced via cell-free expression was purified and its identity was verified using SDS-PAGE analysis. However, the analysis revealed that there was no detectable protein band at the expected size of 13 kDa. For a more comprehensive description of the DBTL process related to this issue, please refer to the engineering section.

CEA N domain expression in cells

Although the CEA N domain was expressed with cell-free expression, a sufficient amount of protein was not obtained to be used in EMSA. This suggests the possibility that by optimizing the experimental conditions of CFE, a sufficient amount of protein can be obtained and synthetic biology techniques such as CFE can be used to confirm the binding affinity between aptamers and biomarkers of other disease, to proceed as quickly as possible with EMSA, which checks the binding of N56 aptamer and CEA N domain, it was intended to express the CEA N domain in the cell.

Therefore, to proceed as quickly as possible with EMSA, which checks the binding of N56 aptamer and CEA N domain, it was intended to express the CEA N domain in the cell.

We conducted protein purification using Ni-NTA nickel beads and Ni-NTA cobalt beads due to a shortage of spin columns. Additionally, as native proteins needed to be purified, we used NaH2PO4 instead of tris for protein purification. The optimization process for CEA N domain protein purification (DBTL process) can be found in the engineering section.

The preparations, protocol, and results of the experiment are as follows.

Preparations (CEA N domain expression in cells)

CEA N domain protein expression

DH10B (negative control)

DH5α DE3 (Sample)

DNA template

Lysogeny broth (LB)

Shaker

Centrifuge 

LB plate

Glass beads

Protein purification

 (pH 8.0)

50 mM NaH2PO4

300 mM NaCl

10 mM Imidazole

 (pH 8.0)

50 mM NaH2PO4

300 mM NaCl

20 mM Imidazole

 (pH 8.0)

50 mM NaH2PO4

300 mM NaCl

100 mM Imidazole

 (pH 8.0)

50 mM NaH2PO4

300 mM NaCl

250 mM Imidazole

 (pH 8.0)

50 mM NaH2PO4

300 mM NaCl

500 mM Imidazole

Lysozyme

Benzonase endonuclease

2 mL tube

1.5 mL tube

Ni-NTA agarose bead

Cobalt resin

Centrifuge

Protein synthesis protocol

CEA N domain protein expression

[Plasmid cloning & CEA N domain expression

1. Add the 80 ng/µL of GOI (gene of interest) in the aliquoted competent cell.
2. Thaw on ice for 20 minutes.
3. Apply heat shock at 42˚C for 45 seconds.
4. Thaw on ice for 2 minutes.
5. Add 800 µL of warm LB on each tube.
6. Vigorously shake samples at 200 rpm, 37˚C at the shaker for 1 hour.
7. Centrifuge the tubes at 13,000 rpm for 1 min at RT.
8. Re-suspend 200 µL of warm LB.
9. Apply 200 µL of the solution on each LB plate (including antibiotics) with glass beads/spreader.
10. After completing step 9, allow one hour to elapse, and then commence measuring optical density (OD) at 15-minute intervals.
11. Incubate each sample for 4 hours at 37˚C, 30˚C, and 16˚C with continuous agitation at 150 rpm. Additionally, include 200 µL of a 1000x IPTG solution in the incubation process.
12. Centrifuge the cells at 4˚C and 3000 rpm for 35 minutes.
13. Discard the flowthrough and retain only the supernatant.
14. To verify the successful expression of the protein, an expression test will be performed.

The chosen strain for protein expression: E. coil (DH5alpha_DE3)


1. Versatility: E. coli is a commonly used host organism for protein expression in research and biotechnology due to its well-characterized genetics and the availability of various expression vectors.

2. Rapid Growth: E. coli has a short generation time, allowing for quick production of protein.

3. Strong Promoters: The DH5alpha_DE3 strain contains a T7 RNA polymerase gene under the control of the lacUV5 promoter, which can be induced by adding IPTG (Isopropyl β-D-1-thiogalactopyranoside). This system enables tight control of protein expression.

4. High Yield: E. coli can produce a high yield of recombinant protein when optimized for the specific protein of interest.

Protein purification

[Ni-NTA agarose bead]

[Lysis]>

1. Thaw the cell samples on ice for 15 minutes. 2. Add 2 mL of lysis buffer to the downed cell and resuspend it. 3. Add 1.4 mL of lysozyme(10 mg/mL) and 1.2µL (250 U/µL) of benzonase endonuclease to the cell sample. 4. Incubate samples on ice for 30 minutes. 5. Centrifuge the samples at 10,000 x g for 45 minutes at 4°C. 6. Take 1.6 mL of lysate by the ratio of Ni-NTA agarose bead. 7. Add 400 µL of Ni-NTA agarose bead to a 2 mL tube and centrifuge at 3,000 rpm for 5 minutes at 4°C. 8. Discard the supernatant, preservation solution. 9. Put 1.5 mL of lysis buffer into a 2 mL tube containing Ni-NTA agarose bead and centrifuge at 3,000 rpm for 5 minutes at 4°C for washing. 10. Discard the supernatant. 11. Repeat step 9. 12. Repeat step 10. 13. Put lysate taken in step 6 into a 2 mL tube containing Ni-NTA agarose bead. **[Binding]** 1. After agitation, shake at 4°C for 1 hour at an appropriate intensity. 2. Centrifuge the samples at 800 x g for 10 minutes at 4°C. 3. Remove the supernatant containing unbound protein for subsequent SDS-PAGE. **[Washing]** 1. Add 1.6 mL of wash buffer to the sample, agitate, and centrifuge at 800 x g for 5 minutes at 4°C. 2. Remove the supernatant, store it separately, and repeat step 17 (1st washing). 3. Remove the supernatant, store it separately, and repeat step 17 (2nd washing). 4. Remove the supernatant as much as possible, and store it separately (3rd washing). **[Elution]** 1. Add 500 µL of 100 mM imidazole elution buffer to the sample. 2. Incubate samples on ice for 10 minutes. 3. Centrifuge the samples at 500 x g for 10 minutes at 4°C. 4. Take the supernatant and add 500 µL of 250 mM imidazole elution buffer to the sample. 5. Repeat steps 22 and 23. 6. Take the supernatant and add 500 µL of 500 mM imidazole elution buffer to the sample. 7. Repeat steps 22 and 23. 8. Take the supernatant. **[Cobalt resin]** **[Lysis]** 1. Thaw the cell samples on ice for 15 minutes. 2. Add 2 mL of lysis buffer to the downed cell and resuspend it. 3. Add 1.4 mL of lysozyme(10 mg/mL) and 1.2µL (250 U/µL) of benzonase endonuclease to the cell sample. 4. Incubate samples on ice for 30 minutes. 5. Centrifuge the samples at 10,000 x g for 45 minutes at 4°C. 6. Take 1.6 mL of lysate by the ratio of cobalt resin. 7. Add 400 µL of cobalt resin to a 2 mL tube and centrifuge at 3,000 rpm for 5 minutes at 4°C. 8. Discard the supernatant, preservation solution. 9. Put 1.5 mL of lysis buffer into a 2 mL tube containing cobalt resin and centrifuge at 3,000 rpm for 5 minutes at 4°C for washing. 10. Discard the supernatant. 11. Repeat step 10. 12. Repeat step 9. 13. Put lysate taken in step 6 into a 2 mL tube containing cobalt resin. **[Binding]** 1. After agitation, shake at 4°C for 1 hour at an appropriate intensity. 2. Centrifuge the samples at 800 x g for 10 minutes at 4°C. 3. Remove the supernatant containing unbound protein for subsequent SDS-PAGE. **[Washing]** 1. Add 1.6 mL of wash buffer to the sample, agitate, and centrifuge at 800 x g for 5 minutes at 4°C. 2. Remove the supernatant, store it separately, and repeat step 17 (1st washing). 3. Remove the supernatant, store it separately, and repeat step 17 (2nd washing). 4. Remove the supernatant as much as possible, and store it separately (3rd washing). **[Elution]** 1. Add 500 µL of 100 mM imidazole elution buffer to the sample. 2. Incubate samples on ice for 10 minutes. 3. Centrifuge the samples at 500 x g for 10 minutes at 4°C. 4. Take the supernatant and add 500 µL of 250 mM imidazole elution buffer to the sample. 5. Repeat steps 22 and 23. 6. Take the supernatant and add 500 µL of 500 mM imidazole elution buffer to the sample. 7. Repeat steps 22 and 23. 8. Take the supernatant.

SDS-PAGE

1. Combine protein sample with 5x sample buffer. Also, prepare a control sample.
2. Incubate at 100℃ for 5 minutes.
3. Load 5 μl of the standard protein marker into the first lane.
4. After a quick microcentrifuge spin, load 5 μl of samples directly onto the 15% gel. To ensure uniform mobility, load an equal volume of SDS-PAGE Blue loading buffer into any unused wells.
5. Run the gel at 80-120V until the protein is completely separated by molecular weight.
6. Stain with Coomassie Blue stain buffer overnight.
7. Destain with destain buffer for overnight.
8. Analyze the location and thickness of the band appearing in the gel.

Results

Protein purification

Most of the proteins that do not bind to the beads are extracted in the unbound step, resulting in the highest protein concentration. As we progress through the washing steps, such as wash 1, wash 2, and wash 3, proteins weakly bound to the beads should be washed away, leading to lower protein concentrations.

In the elution steps, elution 100 (imidazole concentration: 100 mM), elution 250 (imidazole concentration: 250 mM), and elution 500 (imidazole concentration: 500 mM), protein concentrations should increase progressively.

5. EMSA(Electrophoretic Mobility-Shift Assay)

EMSA is an experimental method that can check the degree of binding between genes and proteins. The main principle is to label the DNA probe with radiation isotopes or non-radiation isotopes such as fluorescein to react with proteins at room temperature to determine the degree of band shift in electrophoresis to confirm the binding between proteins and genes.

EMSA was conducted to qualitatively check whether the N56 aptamer is well bound to the CEA N domain and to determine whether the N56 aptamer is appropriate for use in CEApture, LFA-type sensor.

While we were unable to conduct the EMSA experiment before the Wiki freezing, KUAS continues to progress with the experiments. We will endeavor to include any relevant information about the EMSA experiment results in a future presentation video, if possible.

The preparations, protocol[7], and results of the experiment are as follows.

Preparations(EMSA)

**EMSA running buffer (0.5x TBE running buffer)**

10x TBE buffer

Tris base

Boric acid

0.5 M EDTA (pH 8.0)

dH₂O

dH₂O

**Non-denature PAGE gel (6%)**

40% acrylamide

2% bis-acrylamide

10x TBE buffer

TEMED

10% ammonium persulfate

dH₂O

**2x gel shift reaction buffer**

50% glycerol

1 M HEPES (pH 7.9)

1 M Tris-HCl (pH 8.0)

0.5 M EDTA (pH 8.0)

100 mM DTT

dH₂O

BSA (1 µg/µL)

Poly (dI-dC) (0.5 µg/µL)

FAM-labeled N56 DNA aptamer

CEA N domain protein

EMSA protocol

1. Mix 12 μL of 2x reaction buffer, 3 μL of BSA, 2 μL of poly (dI-dC), 3 μL of CEA N domain protein, and 3 μL of dH₂O, then incubate at room temperature or on ice for 30 minutes.
2. Add 1 μL of FAM-labeled N56 DNA aptamer.
3. Keep at room temperature for 20 minutes.
4. Load the gel and run at 200 V for 1 hour to 1 hour and 30 minutes. Use the DNA loading buffer in lane 1 as an indicator of the free probe.
5. Stop the gel when the dye runs at 3 cm to the bottom.
6. Dry the gel and expose the dried gel to X-ray film at -70˚C overnight.
7. Develop the film.

Reference

[1] Gottlieb, H. E., Kotlyar, V., & Nudelman, A. (1997). NMR chemical shifts of common laboratory solvents as trace impurities. Journal of organic chemistry, 62(21), 7512-7515.

[2]  Johanna-Gabriela Walter, Öznur Kökpinar, Karl Friehs, Frank Stahl, and Thomas Scheper Analytical Chemistry 2008 80 (19), 7372-7378 , DOI: 10.1021/ac801081v
      
[3] NEW ENGLAND BioLabs, NEBExpress Cell-Free E. Coli Protein Synthesis System. NEW ENGLAND BioLabs.
      
[4] A. Krop-Watorek, S. Oikawa, Y. Oyama, and H. Nakazato, “Oligomerization of N-terminal domain of carcinoembryonic antigen (CEA) expressed inEscherichia Coli,” Biochemical and Biophysical Research Communications*, vol. 242, no. 1, pp. 79–83, 1998. doi:10.1006/bbrc.1997.7920 
      
[5] D. M. Bollag, M. D. Rozycki, and S. J. Edelstein, Protein Methods. New York: Wiley-Liss, 1996.
      
[6] QIAGEN, Ni-NTA Spin Kit Handbook, 2nd ed. QIAGEN, 2008. https://www.qiagen.com/us/resources/resourcedetail?id=3fc8c76d-6d21-4887-9bf8-f35f78fcc2f2&lang=en
      
[7] J. Y. MD, Gel Shift / EMSA Protocol. Pennington Biomedical Research Center.