Protocols

Materials

• LB broth powder
• MiliQ water

Procedure

Materials

• LB broth powder
• LB agar
• MiliQ water

Procedure

1. Put in 300 mL of MiliQ water, 6 g of LB broth powder, and 4.5 g of LB agar into a bottle. Mix thoroughly, and then autoclave.
2. When pouring plates, microwave the pre-made LB agar for 5 mins to melt.
3. Preparing plates for growing DH5α: add 1 : 100 ratio of X-Gal (20 mg/mL), 1:1000 ratio of IPTG (1 M), and antibiotics (e.g. 1 : 1000 ratio of 34 mg/mL chloramphenicol). Pour 25 mL of the mixture into each labelled plate.
4. Preparing plates for growing BL21: add antibiotics (e.g. 1 : 1000 ratio of 34 mg/mL chloramphenicol). Pour 25 mL of the mixture into each labelled plate.

Materials

• 1 X TAE
• Agarose
• Ladder (7 µL)
• Loading Dye (2 µL used for 10 µL of sample)
• Gel red

Procedure

1. Add 2 g of agarose and 200 mL of 1XTAE into a bottle (1 : 100).
2. Loose the lid of the bottle; use microwave to heat 1 min, shake and heat for another 1 min. The agarose should have dissolved after heating. Use insulated gloves when handling hot bottles.
3. Wait for the solution to cool down. When it is cold enough to be held by hand, add 20 µL of gel red (1 : 10,000).
4. Mix the gel red dye by shaking the bottle slightly and pour the solution on to a mold.
5. Wait until the gel to solidify; transfer the gel to the gel tank and pour sufficient amount of 1XTAE to submerge the gel.
6. Load 7 µL ladder; add 2 µL of purple loading dye into each plasmid sample. After loading, set to 180 V, 400 mA to run.

• Weight 2.381 g IPTG and add 10 mL of sterile water.
• Pass through 0.2 μm filter.
• Aliquot in 2 mL tubes and store at -20°C.

• 0.2 M Tris 24.22 g/L
• 0.5 mM EDTA 0.19 g/L
• 0.5 M sucrose 171.15 g/L
• Adjust the pH to 8.0 with HCl, and then use H2O to make up to 1 liter.
• TES buffer can be stored for months at 4°C.

• 0.1 M Tris 15.76 g/L
• 2.5 M NaCl 146.1 g/L
• 0.15 M imidazole 10.21 g/L

• 0.02 M Tris 3.152 g/L
• 0.06 M imidazole 4.0848 g/L
• 0.5 M NaCl 29.22 g/L

• 0.02 M Tris 3.152 g/L
• 0.5 M NaCl 29.22 g/L
• 0.5 M Imidazole 34.038 g/L

Materials

• 250 ng/mL of template DNA
• 10 μM of forward primer
• 10 μM of reserse primer
• 1x VeriFi Buffer
• 1x VeriMax Enhancer
• 0.02 U/μL high fideilty VeriFi DNA polymerase (PCR Biosysem, UK)

Procedure

1. Make a 50 μL mixture by adding the following reagents into a PCR tube:
   5 X VeriFi Buffer: 10μ L;
   10 X VeriMax Enhancer: 5 μL;
   Forward Primer (10 µM): 2 μL;
   Reverse Primer (10 µM): 2 μL;
   Template DNA: < 10 ng less complex DNA (5 ng);
   VeriFi Hot start polymerase (2 u/μL): 0.5 μL;
   Nuclease-Free Water: add up to 50 μL.
2. Pipette up and down to mix.
3. Put the PCR tube into PCR instrument and set the following program:
   The first denaturation step is 98°C for 30 s.
   Then followed by 20 cycles with:
   • a denaturation step at 94°C for 15 s
   • an annealing step at 65–55°C for 30 s
   • an extension step at 72°C for 38 s
   This is followed by 20 cycles:
   • a denaturation step at 94°C for 15 s
   • an annealing step at 55°C for 30 s
   • an extension step at 72°C for 38 s
   The final extension is performed at 72°C for 2 min.

Materials

• DNA insert
• DNA backbone
• 10x T4 DNA Ligase buffer
• T7 DNA Ligase
• BsaI-HF
• PCR tubes

Procedure

Calculate DNA insert : plasmid volume for 3:1 ratio

1. Add 2 µL of T4 DNA ligase buffer into each PCR tube using.
2. Add diluted plasmid to each PCR tube using.
3. Add specified amount of DNA inserts.
4. Add specified amount of Nuclease-free water.
5. Note: the MiliQ Water should add up the total volume of reaction mixture to 20 µL.
6. Add 1 µL of BsaI HFV2 to each tube.
7. Add 1 µL of T4 ligase to each tube.
8. Incubate reaction mixture in a thermocycler with the following program:
   • 35 cycles, 37 °C for 5 min (Restriction Enzyme);
   • 16°C for 5 min (T4 DNA ligase);
   • then, 65°C for 20 min (Heat inactivation);
   • hold temperature of 4 °C.

Materials

• 5 mL LB agar
• 50 µL chloramphenicol (34 mg/mL)
• 1 colony

Procedure

These steps should be carried out in the hood.

1. Pipette 5 mL of LB medium into each falcon tube.
2. Pipette 5 µL of chloramphenicol into each falcon tube.
3. Place the dishes upside down, pick 1 colony with a hoop. If this is a DH5α culture, pick a white colony (blue-white screening).
4. Insert the hoop into the falcon tube and swirl it until the colony dissolves in the solution.
5. Put the falcon tubes in incubator at 37°C, 220 rpm, overnight.

Materials

• LB Agar Plates containing antibiotics
• SOC Medium
• Competent Cells BL21 (DE3)

Procedure

1. Before starting, equilibrate a water bath to 42°C, warm the vial of SOC medium to room temperature, place the plates in a 37°C incubator to remove excess moisture, and defrost chemically competent cells on ice.
2. Add 5 μL of plasmid DNA to 50 μL of competent cells (1:10 dilution). Mix by tapping, not pipetting.
3. Incubate the vial(s) on ice for 30 minutes.
4. Heat shock: Incubate for exactly 30 s in the 42°C water bath. Do not mix or shake.
5. Remove vial(s) from the 42°C bath and quickly place on ice for 30 s.
6. Add 250 µL of pre-warmed SOC medium to the vial(s).
7. Incubate the vials for 1 hour at 37°C, 250 rpm.
8. Conduct the following cell seeding procedure in a hood.
9. Pipette 100 µL of transformed cells onto each plate; spread the cells evenly with the spreader.
10. Place the LB agar plate overnight at 37˚C. Make sure the plates are placed upside down in the incubator to prevent water from disturbing the colonies.
11. Afterwards, store the plates in a fridge at 4˚C.

Materials

• LB Agar Plates containing antibiotics
• SOC Medium
• Competent Cells BL21 (DE3)

Procedure

1. Before starting, equilibrate a water bath to 42°C, warm the vial of SOC medium to room temperature, place the plates in a 37°C incubator to remove excess moisture, and defrost chemically competent cells on ice.
2. Add 1 μL of plasmid DNA to 25 μL of competent cells (1:25 dilution). Mix by tapping, not pipetting.
3. Incubate the vial(s) on ice for 30 minutes.
4. Heat shock: Incubate for exactly 30 s in the 42°C water bath. Do not mix or shake.
5. Remove vial(s) from the 42°C bath and quickly place on ice for 30 s.
6. Add 250 µL of pre-warmed SOC medium to the vial(s).
7. Incubate the vials for 1 hour at 37°C, 250 rpm.
8. Conduct the following cell seeding procedure in a hood.

Materials

• Autoclaved LB media
• Autoclaved baffled shake flask
• Antibiotics: Chloramphenicol (34 mg/mL)
• IPTG (1 M)
• TES buffer (pH 8.0)

Procedure

Protein Expression Day 1

1. Measure the OD at 600 nm of the overnight starter culture of BL21 by using a P1000 pipette to take a 1 mL sample.
2. Calculate the volume needed to dilute the OD to 0.05 in 200 mL LB media.
3. Inoculate the shake flask with the overnight culture and incubate the shake flask at 37°C, 250 rpm.
4. After 90 minutes, measure the OD at 600 nm. Keep measuring the OD at regular intervals (e.g., every 30 minutes).
5. When the OD reaches between 0.6 and 0.8, induce with IPTG 1 mM (1:1000 dilution) and note down the time. For a 200 mL culture, add 200 µL of sterile 1 M IPTG.
6. Incubate for 16-20 hours at 25°C and 250 rpm.

Protein Expression Day 2

1. If needed, take 500 µL of the sample, and boil to get a crude extract for SDS-PAGE analysis. Collect the bacteria by centrifuging for 15 minutes at 9,000 g at room temperature.
2. Discard the supernatant, and then carefully resuspend the cell pellet in 3 mL of TES buffer.
3. Incubate the cell suspension for at least 1 hour on ice on an orbital shaking platform.
4. Add 6 mL of pre-chilled sterile MilliQ water and incubate the mixture on ice on an orbital shaking platform for 45 minutes.
5. If needed, take a 500 µL sample of the cell resuspension for SDS-PAGE analysis. Centrifuge the suspension for 30 minutes at 10,000 g at 4°C and recover the supernatant as the periplasmic extract.
6. If needed, take a 500 µL sample of the supernatant for SDS-PAGE analysis.

Materials

• 5 X Binding Buffer (pH 7.4)
• 1 X Wash Buffer (pH 7.4)
• 1 X Elution Buffer (pH 7.4)
• 0.22 µm filter and syringe
• HIS-tag chromatography column
• Resin
• 0.1 M nickel salt solution (NiSO4)
• Column

Proceduce

1. Equilibrate the sample by adding 200 mL of sample with 50 mL of 5X binding buffer.
2. Assemble the column according to the gravity column assembling protocol.

Blank Run

1. Wash Column with 5 column volumes of MiliQ water.
2. Equilibrate with 10 column volumes of 1x binding buffer.

Purification

1. Pass the prepared sample through the column.
2. Collect sample flowthrough for SDS-PAGE analysis.
3. Equilibrate with 10 column volumes of 1X binding buffer.
4. Collect the binding buffer flowthrough for SDS-PAGE analysis.
5. Run 5 column volumes of 1X wash buffer.

Elution

1. Run 5 column volumes of 1 X elution buffer, and collect as 2 mL aliquots.

Cleaning

1. Wash with 10 column volumes of MiliQ water.
2. Wash with 5 column volumes of 20% EtOH and store the columns and the tubing at 20% ethanol in 4°C.

Materials

• Vivaspin 20 with a 30 kDa cutoff
• Samples to be buffer exchanged
• 5X Binding Buffer (pH 7.4)

Procedure

1. Pour all samples into Vivaspin 20.
2. Spin the tubes for 15 mins at 5000g.
3. Centrifuge the tubes for 5 minutes if the remaining liquid is more than 5 mL.
4. Resuspend the retained liquid with 10 mL of binding buffer (the new buffer).
5. Remove all liquid from the Vivaspin and aliquot into 2 mL Eppendorf tubes for later uses.
6. Store the Eppendorf tubes at 4°C.

Materials

• 5 mL LB agar.
• 5 µL kanamycin.
• 1 scrape of Tinsel Purple glycerol stock.

Procedure

1. Pipette 5 mL of LB medium into each Falcon tube.
2. Pipette 5 µL of kanamycin into each Falcon tube.
3. Take a scrape of the Tinsel Purple glycerol stock with a hoop.
4. Insert the hoop into the Falcon tube and swirl it until the colony dissolves in the solution.
5. Put the Falcon tubes in the incubator at 37°C and 220 rpm overnight.

Materials

Procedure

1. Collect the bacteria by centrifugation for 15 min at 9,000 g at room temperature.
2. Discard the supernatant, then carefully resuspend the cell pellet of 1 L of culture in 15 mL of ice-cold Tris NaCl buffer.
3. Sonicate the mixture for 20 cycles with a 10-second resting interval.
4. Centrifuge again at 4,000 g at 4°C for 30 minutes.
5. Collect the supernatant and dispose of the sediment, because the proteins are now in the supernatant.

Materials

• Equilibration buffer: 20 mM Tris-Cl pH 8.5 with 25 mM NaCl
• Elution buffer: 20 mM Tris-Cl pH 8.5 with 350 mM NaCl
• High Salt Wash: 20 mM Tris-Cl pH 8.5 + 1 M NaCl
• Empty chromatography column (10 mL)
• Q Sepharose
• 20% Ethanol

Procedure

1. Mix Q Sepharose well.
2. Add a filter to your column and push it down to the bottom of the column using a pen; the little filter has a smooth and a rough side, the rough side faces upwards.
3. Pipette 2.5 mL of resin into the column.
4. Cut the bottom of the column and let the ethanol out.
5. Add 5 CV water to wash ethanol out.
6. Store the column with ethanol before usage.
7. To use, equilibrate with your buffer and run your protocol.

1. Equilibrate the column with 5 CV equilibration buffer.
2. Load sample and collect the flow through.
3. Add 5 CV of equilibration buffer to wash off loosely binding proteins; collect the flow through in a Falcon tube.
4. Add 5 CV of elution buffer to elute Tinsel Purple; collect the flow through in 5 small 2 mL Eppendorf tubes.
5. Add 5 CV of high salt wash to wash off the tightly bound proteins so the column is clean for reuse; collect the flow through in a Falcon tube.

Materials

For diagnostic digest:

• Plasmid to be tested (5µL)
• Restriction enzyme 1 (0.5µL)
• Restriction enzyme 2 (0.5µL)
• enzyme buffer (1µL)
• MilliQ water (3µL)

Procedure

1. Add plasmids, MilliQ water, enzyme buffer and enzymes in the stated order.
2. Once all added, place the mixtures in a 37 °C water bath for 15mins.
3. Add another 0.5µL of each enzyme into the mixture.
4. Incubate in 37 °C water bath for 45 minutes.
5. Give the mixture a short centrifuge to remove condensation.
6. Add in 2 µL of 6x loading dye.
7. (skip if gel is ready) If agarose gel has not been prepared, store samples at -20C.
8. All samples (12 µL) into the wells.

Materials

• Loading dye
• 2-mercaptoethanol/ Dithiothreitol (DTT)
• Molecular weight ladder
• 10x SDS running buffer
• 10x SDS running buffer
• Gels
• InstantBlue

Proceduce

Sample preparation

1. Mix samples (1-2 µg if purified protein) with loading dye. Dilute with MilliQ water to reach appropriate loading dye concentration.
2. Optional: add reducing agent (e.g. 2-mercaptoethanol/ Dithiothreitol (DTT)) to disrupt disulphide bonds present in protein.
3. Boil samples for 10 min at 95°C. Tubes lids may pop off - this can be avoided by piercing a hole in the lid with a needle. Centrifuge samples briefly.

Set up the gel tank

1. Dilute 10x SDS running buffer in MilliQ water to achieve 1x solution (e.g. 100 mL of 10x SDS running buffer with 900 mL MiliQ water).
2. Place the gel inside the gel tank with the appropriate inserts. If using commercial gels, make sure to remove the comb and any strips of tape at the bottom of the gel (otherwise it will not run properly). If only running one gel, use another gel/ support to prevent leaking.
3. Optional: wash wells three times with running buffer using a Pasteur pipette.
4. Add 1x SDS running buffer between the gels until the wells are covered. Wait for 10 s to see if any liquid leaks from between the gels. If not, proceed to fill the outer chamber with 1x SDS running buffer. If buffer leakage between the gels occurs, reposition the gels inside the insert and repeat.

Loading the gel

1. Load appropriate volume of sample into each well. Leave at least one well empty for the addition of molecular weight ladder. Optional: add 1x loading dye to any unused wells to improve the running of the gel (i.e. avoid 'smiling bands').
2. Place lid on the gel tank and run the gel at the recommended voltage using the power supply based on manufacturer’s specifications (200 V for 50 minutes is typical). Run until the loading dye is just before the gel front.

Coomassie staining the gel

1. Once the loading dye has reached the gel front, stop the power supply.
2. Remove the gel from the casing using the gel knife (careful not to tear the gel).
3. Add 10 mL of InstantBlue into one of the large weighing boats and place the gel inside. Add the other weighing boat on top to act as a lid to avoid evaporation.
4. Incubate the gel with the InstantBlue for at least 1 h (or overnight to achieve maximal staining) and then wash with water to wash the gel. Image the gel.

Materials

• Trans-blot turbo PVDF/ Nitrocellulose transfer packs
• Primary antibody
• Secondary conjugated antibody (this is unnecessary if your primary antibody is conjugated to a detection enzyme)
• TBST (50 mM Tris-HCl, 150 mM NaCl and 0.1% Tween-20, topped up to desired concentration using MilliQ water)
• Blocking solution: 5% milk powder or 2-5% bovine serum albumin (BSA) in 1x TBST or PBST
• Detection reagents (this depends on detection enzymes conjugated to antibodies). Note: If fluorescence conjugation is used no detection reagents may be required

Proceduce

Transferring protein bands to membrane

1. Once gel is removed from casting, carefully cut off wells with the gel knife.
2. Open Transblot turbo transfer pack and place the stack containing the membrane on the bottom of the loading cassette tray. Try not to touch the membrane. Carefully lay the gel onto the membrane.
3. Using the gel knife to cut off the bottom ridge of the gel and remove with tweezers.
4. Use the roller to remove any bubbles in between the gel and the membrane.
5. Place the second stack on top of the gel. Use the roller to flatten this and remove any bubbles.
6. Place and secure lid (by turning green handle to lock) and slide into Trans-blot apparatus.
7. Select 'Turbo' setting and then the appropriate gel size and then press run for 'A' or 'B' depending on where you have placed your cassette.

Blocking

1. When Trans-blot Turbo run has finished carefully remove membrane with tweezers and place into large weighing boat. Use a second weighing boat to cover.
2. Cover membrane in blocking solution and leave on shaker for 1hr at RT or overnight at 4ºC.

Primary antibody addition

1. Dilute primary antibody solution according to manufacturer instruction in blocking solution Milk/BSA-TBST/PBST. 5 mL will suffice for a mini-blot but as little as 3mL can be used if antibody stock is low. Vortex to mix well.
2. Pour off blocking solution from previous step and replace with primary antibody solution.
3. Place on shaker for 1hr at RT or overnight at 4ºC.

Secondary antibody addition (if primary antibody conjugated move straight to 'development')

1. Wash membrane 3x for 5 minutes with TBST/ PBST. Place on shaker for each incubation step.
2. After incubation prepare secondary antibody solution using the same method as for primary antibody. Dilutions will likely be higher and may need some troubleshooting depending on your chosen antibody. Place on shaker for 1hr at RT.

Development

1. Wash membrane 3x for 5 min with TBST/PBST. Place on a shaker for each incubation step.
2. Development will be different depending on conjugated antibody. Follow manufacturers instruction.
3. Fluorescence and Chemiluminescence blots can be viewed on the gel doc in lab 2.19. Ask lab managers for training.
4. Some developments can be visualized by eye. In this case stop the development when suitable bands are observed. Dry the blot on blue roll and scan blot once dried.

Materials

• Bovine Serum Albumin
• Unknown concentration of protein sample
• 96-well plate
• plate reader

Procedure

1. Plan the arrangement of samples and dilution series of BSA on a 96-well plate.
2. Prepare the following dilution series of BSA. The stock BSA concentration is 0.1 mg/mL.

Protein Concentration (µg/mL)	Protein Standard Stock Volume (µL)	Water (µL)	Total Volume (µL)
80	160	40	200
50	100	100	200
30	60	140	200
25	50	150	200
20	40	160	200
15	30	170	200
10	20	180	200
5	10	190	200
0	0	200	200

3. Dilute the unknow protein elution flowthrough sample in water (1:4) to avoid background noise of imidazole in the elution buffer.
4. Add 5 µL of the known dilutions of BSA with 250 µL of Bradford dye reagent. Repeat the same dilution three times.
5. Add 5 µL of the diluted unknown protein sample with 250 µL of Bradford dye reagent.
6. Add 255 µL of Bradford dye reagent as a blank.
7. Put the 96-well plate into a plate reader and set the wavelength to 595 nm. The absorbance value is then plotted on a graph against the BSA concentration to obtain a standard BSA concentration curve. Any sample with unknown concentration can then be determined using the curve.

Materials

• 1X Binding buffer
• Stock of fusion protein
• Black clear-well 96-well plates (flat bottom)
• Foil
• NanoGlo luciferase assay

Procedure

1. Prepare a series dilution of fusion proteins. For example, prepare the following for NanoLuc and nanobody-NanoLuc: 1 µg/mL, 3 µg/mL, 5 µg/mL, 10 µg/mL. For controls, pipette 100 µL of NanoGlo with 100 µL of binding buffer.
2. Prepare NanoGlo by adding furimazine into NanoGlo buffer in a 1:50 ratio.
3. Pipette 100 µL of diluted fusion protein and 100 µL of NanoGlo per well.
4. Set the plate reader to read bioluminescence at 470-480 nm for 30 min to 1 h, with 30 s intervals between each reading. Cover the plate with foil and start measurement immediately.

1. Prepare a series dilution of fusion proteins.
2. Pipette 100 µL of diluted fusion protein into each well as planned. Then, pipette 100 µL of 1X binding buffer.
3. Set the plate reader to read absorbance at 588 nm (endpoint reading).

Materials

• 96-well flat, clear-bottomed black plates
• Cystatin C biomarker (Proteintech)
• PBS
• Tween-20
• MiliQ water
• Tween-20
• Bovine serum albumin (BSA)
• Paper towels
• Nanobody Conjuagtes Stock

Proceduce

1. Prepare PBS-0.05% Tween-20 (e.g. 0.1 mL Tween-20 to 199.9 mL PBS)
2. Prepare blocking buffer: 3% BSA in PBS (e.g. 0.45g of BSA, 14.55 mL of PBS)
3. Prepare a series of diluted biomarker , Cystatin-C, in PBS as coating solution. For example, we prepared the following concentration: 1000 ng/mL, 100 ng/mL, 10 ng/mL, 1 ng/mL, and 0.1 ng/mL.

1. Pipette 50 µL of appropriate coating solution per well as planed.
2. Put on plate rocker for 1h.
3. Remove coating solution and tap plate on a paper towel to dry thoroughly.
4. Wash each well three times with 300 µL of 1X PBS-0.05% Tween-20. After each wash, remove wash solution and tap towel on to dry. Use multi-channel pipette to speed up the process.

1. Add 100 µL of blocking buffer. Incubate for 1.5h at room temperature on a plate shaker.
2. Remove the solution and wash each well with 300 µL of wash buffer 1X PBS-0.05% Tween-20 3 times.

All conjugates are diluted in blocking buffer into the following concentrations: Nanobody-NanoLuc 1 × 10 ^-8 M, Nanobody-NanoLuc-NanoLuc 5 X 10^-9 M, Nanobody-tinsel Purple 1.12 X 10 ^-6 M, tinsel Purple 1.12 X 10^-6 M

1. Add 100 µL of conjugates per well and shake for 1.5h.
2. Wash five times with 300 µL PBS-0.05% Tween-20.

• For NanoLuc related conjuagtes: Prepare NanoGlo by adding 5000 µL of NanoGlo buffer to 100 µL of furimazine. Add 100 µL of NanoGlo per well. For negative control without conjuagte or well with only biomarkers, add 100 µL of 1X PBS to ensure an euqal overall volume. Cover the plate with a foil and set the plate reader to read NanoLuc bioluminescence (470-480nm) from the bottom. Read at 30 second intervals for 30 minute to 1 hour.
• For chromoprotein related conjugates: add 100 µL of 1X PBS to each well and set plate reader to read endpoint absorbance at 588 nm.