Experiments
We were fortunate enough to have two full months to optimise our protocols and design our experiments. This provided us with a solid foundation on which to base our proof-of-concept. Below, you can find the experiments we performed, from cloning through to bioluminescence detection, and their corresponding protocols.
Construct design and cloning Protein expression and purification Assay development References

Construct design and cloning

Two fusion proteins of an anchor peptide with the ability to bind polypropylene (PP) together with enhanced green fluorescent protein (eGFP) and NanoLuc luciferase (NLuc) respectively were created to ease detection and quantification of anchor peptides bound to PP. A spacer sequence of 10 alanines, a TEV protease cleavage site, an Avi tag and another spacer sequence of 5 alanines separates eGFP or NLuc from the anchor peptide sterically. The TEV-protease cleavage site allows for the isolation and purification of the anchor peptide (without eGFP or NLuc) for the sandwich bead assay as described below. The plasmid pET45b served as vector backbone for the two different fusion constructs. The sequences for the anchor peptide and the spacer sequence were obtained from Rübsam et al. (2017) and were later codon optimised for E. coli. The insert was cloned into pET45b using restriction enzymes, HindIII and SacI, and restriction cloning. The full construct was transformed into E. coli Top10 cells and the success of the cloning was verified by sequencing.

Protein expression and purification

The fusion proteins were expressed in E. coli BL21 cells and sonication was used for cell lysis. Purification was performed using chromatography with batch purification and Äkta protocols. The fusion proteins were stored in a 1x PBS buffer at 4°C until further usage. Protein concentrations were determined with photometer (280 nm) and protein homogeneity was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE). Western blot was performed to confirm the presence of the target protein. One third of the purified proteins were digested with TEV protease to separate the anchor peptide from the eGFP and NLuc respectively. Chromatography was used to purify the anchor peptide. The cleaved eGFP and NLuc were kept to serve as a negative control for binding. The AviTag in our construct designs was included to enable targeted biotinylation to the magnetic beads. We produced the BirA enzyme from glycerol stocks to facilitate biotinylation. However, due to time constraints we were not able to perform the biotinylation itself.

Assay development

The eGFP and NLuc fusion proteins were tested on polypropolene surfaces to confirm slective binding to polypropolene. For eGFP, this involved the incubation of our eGFP fusion protein in a black polypropolene 96 well plate. The fluorescence intensity was measured with a plate reader prior to and post washing with 1x PBS buffer. For NLuc, we used the Promega Nano-Glo® Luciferase Assay. Our NLuc fusion protein was incubated in a black polypropolene 96 well plate. Follwing the addition of the NLuc substrate and washing 1x PBS, luminescence was measured. The same assay was performed on a polystyrene plate and the differences in luminescence intensity were observed.

Figure showing our NLuc binding assay in a polypropolene plate. Imaged with a smart phone camera.

References

Rübsam, K., Stomps, B., Böker, A., Jakob, F., & Schwaneberg, U. (2017). Anchor peptides: A green and versatile method for polypropylene functionalization. Polymer, 116, 124-132.

Restriction digestion process is used to precisely cleave a DNA fragment at a defined location using restriction enzymes. Upon cleavage at the recognition sites, there is the formation of 'sticky ends' which aids in downstream ligation. The use of two different restriction enzymes at the respective ends of insert and plasmid backbone ensure that the insert is introduced into the vector in the desired orientation.

Materials
  • Nuclease-free water (MiliQ)
  • 10x FastDigest Buffer
  • FastDigest Enzymes Hind III HF and Sac I HF
  • Plasmid DNA pET45b
Procedure
  1. Thaw enzymes and FastDigest Buffer on ice.
  2. Add the following reagents in the following order:
    3. Component Amount
    Water, Nuclease-Free Adjust to a final volume of 20 μl
    10x FastDigest Buffer 2 μl
    Plasmid DNA 1 μg (0.5 μl to 10 μl depending onthe DNA concentration)
    Hind III HF 1 μl
    Sac I HF 1 μl
    Total volume 20 μl
  3. Mix gently and spin down.
  4. Incubate at 37°C in a heat block for 1-2 h.
  5. Optional, inactivate the enzymes by incubating for 20 min at 80°C.

To purify, separate and identify DNA fragments, agarose gel electrophoresis is performed.

Materials
  • Tris base
  • Acetic acid
  • EDTA
  • dH2O
  • Agarose
  • GelRed
  • 1x loading dye
  • DNA ladder
Procedure
  1. For 50x TAE Buffer, mix the following:
    2. Component Amount
    Tris base 242 g (in water)
    Acetic acid 57.1 ml
    EDTA 100 ml
    dH2O Fill up to 1 l
  2. For 1x TAE buffer, add 40 ml of 50x TAE buffer to a measuring cylinder and dilute with dH2O to make it upto 2 L in a glass container.
  3. Gel preparation:
    4. Component Amount
    1% Agarose 1 g / 2 g
    1x TAE buffer 100 ml / 200 ml
  4. Mix the agarose with buffer in a conical flask and heat till the agarose melts and the solution turns transparent. Be careful not to burn yourself as the flask may get too hot. OBS! boiling delay
  5. Add 10 μl / 20 μl of GelRed into this agarose solution and swirl lightly.
  6. Cast the agarose gel on a cast and place the comb on the top part of the gel. Let the gel solidify at room temperature for 20-30 min. Then place the gel onto the electrophoresis apparatus ensuring that it is totally submerged in 1x TAE buffer.
  7. Meanwhile, prepare your DNA samples by adding 1X loading dye solution to the samples (1:1). For example: 1 μl of loading dye into 1 μl of DNA sample.
  8. Carefully load each sample into its designated wells. To determine the DNA fragment sizes on the gel, load a DNA Ladder into one of the wells (5 μl).
  9. Run an electric current of 180 V for 30 minutes.
  10. Visualize gel using a gel reader or under UV light.

To extract DNA fragments from the agarose gel, visible bands are cut out on an UV transilluminator. The QIAGEN Gel extraction and PCR Clean-up Kit is used to extract the DNA according to manufacturer's specifications.

Materials
  • QIAGEN Gel extraction and PCR clean-up kit
  • Isopropanol
Procedure
  1. Excise the DNA fragment from the agarose gel with a scalpel (Make sure to wear protective gear while working in UV)
  2. Weigh the empty tube.
  3. Weigh the gel slice in a tube.
  4. Add 3 volumes of QG Buffer to the gel slice tube - 100 mg ~ 100 μl
  5. Incubate at 50°C for 10 mins until the gel has completely dissolved. Vortex every 2-3 mins to dissolve the gel. The colour of the solution should be yellow.
  6. Add 1 gel volume Isopropanol to this mixture and mix.
  7. Place a QIAguick spin column in a 2 ml collection tube. Add the sample mixture to this column and centrifuge for 1 minute (13000rpm).
  8. Discard the flowthrough and place the QIAguick column back in the same tube. (Sample volume should be less than 800 μl)
  9. Keep repeating the previous step until all of the sample has been used.
  10. Add 500 μl of QG buffer to the column and centrifuge for 1 minute. Discard the flow-through and place the column back in the same tube.
  11. Washing - add 750 μl of PE buffer to the column. Let the column stand for 2-5 minutes.
  12. Centrifuge for 1 minute and discard the flowthrough and place the column back into the same tube.
  13. Centrifuge the column containing the tube once again for 1 minute to remove the residual wash buffer.
  14. Place the column in a 1.5 μl microcentrifuge tube and add 30 μl EB Buffer to the centre of the column.
  15. Let the column stand for 4 minutes and centrifuge for 1 minute.
  16. Measure the DNA concentration using NanoDrop or Qubit.

To combine the vector DNA with the insert DNA, T4 DNA ligase is used to link the fragments. The T4 ligase connects the corresponding sticky ends to create the new plasmid. For the ligation, 100 ng of vector DNA were used, the mass of insert was calculated using the following formula (e.g. using NEBcalculator): minsert = bpinsert/bpvector ∙ mvector∙ 5/1

Materials
  • Linear vector DNA (digested)
  • Insert DNA (digested)
  • 10x T4 DNA Ligase buffer
  • T4 DNA Ligase
  • Nuclease free water
Procedure
  1. Prepare the following reaction mixture:
    2. Component Amount
    Linear vector DNA 20-100 ng
    Insert DNA 1:1 to 5:1 molar ratio (insert:vector)
    10x T4 DNA ligase buffer 2 μl
    T4 DNA ligase 1 μl
    Nuclease free water to 20 μl
    Total volume 20 μl
  2. Incubate for at least 10-20 minutes at 22 °C (room temperature).
  3. Use 8 μl of the mixture for transformation of 10 μl of competent cells.

We used two E. coli strains for our experiments: Top10 and BL21. This protocol outlines how Top10 was transformed with the plasmid of interest for amplification. The protocol for BL21 can be seen below.

Materials
  • 5x KCM stock (0.5 M KCl, 0.15 M CaCl2, 0.25 M MgCl2)
  • Ligation mix
  • 1 aliquot competent cells
  • TSB media
Procedure
  1. Mix 8 μl Ligation mix and 2 μl 5x KCM in a sterile Eppendorf tube.
  2. Chill on ice for 2-5 minutes.
  3. Add 10 μl competent cells.
  4. Incubate on ice for 20 minutes.
  5. Incubate at room temperature for 10 minutes.
  6. Heat shock: 60 sec at 42°C, then incubate for 2 min on ice.
  7. Add 200 μl preheated TSB media (37°C), phenotype on rotamixer 150 rpm at 37 ̊C for 1 hour.
  8. Centrifuge for 3 min 3000 rpm, discard the supernatant and resuspend the pellet in 200 μL TSB.
  9. Plate the 200 μl on agar plate with appropriate antibiotic.
  10. Incubate at 37 ̊C overnight or at room temperature over the weekend.

To purify the amplified plasmid of interest, miniprep is performed. The protocol is adapted from the manufacturers.

Materials
  • QIAGEN Miniprep kit
Procedure
  1. Pellet 1-5 ml bacterial overnight culture by centrifugation at 4600 rpm for 5 min.
  2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a 1.5 mL Eppendorf tube.
  3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed formore than 5 min.
  4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
  6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 60 s and discard the flow-through.
  7. Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30-60 s and discard the flow- through.
  8. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 60 s and discard the flow- through.
  9. Centrifuge for 1 min to remove residual wash buffer.
  10. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 30 μl Buffer EB (10 mM TrisCl, pH 8.5) to the center of the QIAprep 2.0 spin column, let it stand for 4 min, and centrifuge for 1 min.
  11. Load the flowthrough on the column and incubate for 4 min before centrifugate for 1 min.
  12. Determine concentration with NanoDrop.
  13. Send it for sequencing.

The E. coli strain BL21 was transformed for the expression of the protein of interest.

Materials
  • 5x KCM stock (0.5 M KCl, 0.15 M CaCl2, 0.25 M MgCl2)
  • Ligation mix
  • 1 aliquot competent cells
  • TSB media
Procedure
  1. Heat TSB to 37°C and let cell aliquots thaw on ice.
  2. Gently mix the cell aliquots and pipettes 50 μL of cells to each tube.
  3. Add 10 μl of KCM to each tube.
  4. Incubate the cells on ice for 10 mins.
  5. Add 2-5 μL of plasmid and mix gently.
  6. Incubate on ice for 30 mins.
  7. Heat Shock: Heat-pulse for 15 sec, 42°C.
  8. Incubate on ice for 2 mins.
  9. Add 100 μl of per-heated TSB to each tube.
  10. Incubate the cells in shaking incubator for 1 h at 37°C, 200 rpm and preheated plates with relevant antibiotic in the incubator.
  11. Mix and plate it on the agar plate and spread it evenly.
  12. Incubate overnight (12-17 h) at 37°C.

The target protein is expressed in E. coli BL21 cells.

Materials
  • Transformed E. coli BL21
  • TBS media supplemented with ampicillin
  • IPTG (0.5 M)
  • Lysis buffer
Procedure

    Pre-culture
  1. Pick a colony and inoculate in 10 mL of TSB Media with 10 μL Amp in a 50 mL falcon tube overnight at 37°C in a shaking incubator.

    2. Main culture
  2. Centrifuge cell culture at 4600 rpm for 10 min. Discard supernatant and resuspend pellet in 10 mL TSB + 10 μl Amp (1:1000).
  3. Inoculate 200 mL TSB + 200 ul Amp (1:1000) with 4 mL cell suspension (1:50).
  4. Incubation for 2-4 h at 37°C shaking (220 rpm) until OD600 is 0.3-0.6 (then take a sample - 1mL - to be processed!)
  5. ((Glycerol stock: 900 μl of the preculture and 300 μl of 100% glycerol store at -80°C))
  6. Add IPTG (final concentration:1 mM)
  7. Incubate 3 h (37°C, 220 rpm) or overnight at 25°C.
  8. Take a sample (1 mL - to be processed)
  9. Transfer the rest of the sample to GSA tubes or fresh falcon tubes. Centrifuge at 4600 rpm (2800 g) and 4°C for 15 min.
  10. (Discard supernatant and store pellet at -80°C or) continue with cell lysis

    11. Cell lysis by sonication
  11. Resuspend the pellet in 10 mL (batch purification) equilibration buffer and transfer to SS34 tubes (50 mL)
  12. Disrupted by sonication on ice, repeat twice:
    1. First round 1:30 min, interval 1 s, 23% amplitude
    2. Second round 3:00 min, interval 1 s, 23% amplitude
  13. Make sure that the sample is not over-heated. To ensure proper lysis, sonicate all samples once (storing them on ice at all times to cool before and after sonication), and then sonicate each sample again. Wash sonicator with EtOH in between each sample-run. Clean sonicator gently with fine sand paper afterwards.
  14. Spin at 25 000xg, 15 min, 4°C (keep the supernatant!)
  15. Filter the supernatant through a 0.45μm filter.
  16. Take a 30 μl aliquot of the filtered supernatant for SDS-gel analysis, and store at -20°C. Also dip a tip in the pellet and dissolve in 30 μL MilliQ, as a sample for SDS-PAGE of the pellet fraction post-lysis.
  17. Run SDS-PAGE and western blot to confirm expression.

Batch purification of our His6-tagged proteins was carried out. This is protocol is for 200 ml cultures and should be scaled for other volumes.

Materials
  • Pellet of 100-mL production, in SS34 tube
  • Equilibration buffer (optional)
    • 20 mM Tris-HCl, 300 mM NaCl, 10 mM Imidazole, pH 8.0. Filtered 0.2 μm.
    • 40-60 ml per construct (20ml if washing once for equilibration, 60ml if twice)
  • Wash buffer (it can be used also for equilibration)
    • 20 mM Tris-HCl, 300 mM NaCl, 30 mM Imidazole, pH 8.0. Filtered 0.2 μm.
    • 60 ml per construct
  • Elution buffer
    • 20 mM Tris-HCl, 300 mM NaCl, 500 mM Imidazole, pH 8.0. Filtered 0.2 μm.
    • 2.5 ml per construct
  • PD10 columns
  • Goal buffer: ca. 30ml per construct
Procedure

    Buffer preparation:
  1. Equilibration buffer:
    2. Component Concentration (M) Mw (g/mol) Volume (l) Mass (g)
    Tris-HCl 0.02 157.60 0.5 1.576
    NaCl 0.3 58.44 0.5 8.766
    Imidazole 0.01 68.08 0.5 0.3404
  2. Wash buffer:
    3. Component Concentration (M) Mw (g/mol) Volume (l) Mass (g)
    Tris-HCl 0.02 157.60 1 3.152
    NaCl 0.3 58.44 1 17.532
    Imidazole 0.03 68.08 1 2.0424
  3. Elution buffer:
    4. Component Concentration (M) Mw (g/mol) Volume (l) Mass (g)
    Tris-HCl 0.02 157.60 0.5 1.576
    NaCl 0.3 58.44 0.5 8.766
    Imidazole 0.5 68.08 0.5 17.2

    Equilibration of cobalt resin
  4. Thoroughly resuspend the Cobalt Resin.
  5. Leave at room temperature to equilibrate for 15 minutes, followed by thorough resuspension, to ensure a homogeneous solution. This keeps the ratio of resin to slurry at 1:2.
  6. Immediately transfer 4 ml slurry to a 50 ml Falcon tube. Centrifuge at 1000 x g for 1 min and remove supernatant. This corresponds to a CV of 2 mL resin. Supernatant removal is best done by decisive decantation (i.e. quick turning of the tube, with one extra flick).
  7. Wash with 10 CV equilibration Buffer (or washing buffer)

    8. Sample application
  8. Resuspend the Cobalt Resin in the pulp free E. coli juice.
  9. Gently agitate in cold room for 30 min on a rotamixer to allow the his-tagged protein to bind the resin.
  10. Centrifuge at 1000 x g for 1 min.
  11. Carefully remove as much supernatant as possible without disturbing the resin pellet. Note: take a 30 uL sample as flow-through for SDS-PAGE analysis.

    12. Washing
  12. Wash the resin by adding 10 CV of Wash Buffer. Gently agitate the suspension in cold room for 5 min on a rotamixer for thorough washing.
  13. Centrifuge at 1000 x g for 1 min. Remove and discard the supernatant. Take a 30 uL sample of the supernatant and save for SDS-PAGE analysis.
  14. Repeat 12-13 for a total of three washes.

    15. Elution
  15. Elute the his-tagged protein by adding 2.5 ml elution buffer to the resin and incubate for 10 min in the cold room.
  16. Centrifuge at 1000 x g for 1 min. Add the 2.5 ml supernatant to PD10 for buffer exchange.

    17. PD10 column buffer exchange
  17. Remove the bottom and cap off new columns.
  18. Let the buffer flow through.
  19. Equilibrate the column with 25 ml of the goal buffer.
  20. Add exactly 2.5 ml of the sample. Do not collect the first 2.5 ml flow through as the sample enters the column.
  21. When the sample has completely entered the column, switch to 15 ml collection tubes at the bottom of the column.
  22. Add exactly 3.5 ml goal buffer, collect the flow through, which contains the sample exchanged into the new buffer.
  23. Measure A280 and run SDS-PAGE analysis.

Protocol for purification of His6-tagged constructs.

Materials
  • 20ml of clarified cell lysate
  • Wash buffer
    • 20 mM Tris-HCl, 300 mM NaCl, 30 mM Imidazole, pH 8.0. Filtered 0.2 μm.
  • Elution buffer
    • 20 mM Tris-HCl, 300 mM NaCl, 500 mM Imidazole, pH 8.0. Filtered 0.2 μm.
  • MilliQ
  • 70% ethanol
  • ÄKTA:Start
  • Frac30 Fraction Collector
  • Cytiva HisTrap FF Crude (1ml) (or other appropriate ÄKTA column)
Procedure

    Buffer preparation:
  1. Wash buffer:

    Concentration of imidazole in wash buffer must be empirically determined from construct to construct to ensure high purity and high yield.

    2. Component Concentration (M) Mw (g/mol) Volume (l) Mass (g)
    Tris-HCl 0.02 157.60 1 3.152
    NaCl 0.3 58.44 1 17.532
    Imidazole 0.03 68.08 1 2.0424
  2. Elution buffer:
    3. Component Concentration (M) Mw (g/mol) Volume (l) Mass (g)
    Tris-HCl 0.02 157.60 0.5 1.576
    NaCl 0.3 58.44 0.5 8.766
    Imidazole 0.5 68.08 0.5 17.2

    Programme set-up and column connection
  3. Power on the ÄKTA:start system and open the UNICORN software on the connected computer.
  4. Connect the UNICORN software to the ÄKTA:start.
  5. Place the wash buffer, elution buffer, MilliQ water, and 70% Ethanol on top of the machine and immerse inlet tubes A and B into the Ethanol flask.
  6. Place a waste collection flask next to the machine and place all waste outlet tubes into it.
  7. Initiate a manual run with a slow flow rate (0.5ml/min).
  8. Disconnect the G5 connector from the coupling, and connect the tubing to the HisTrap column in a drop-to-drop manner, ensuring no airbubbles are introduced.
  9. Disconnect the G6 connector from the coupling and connect the HisTrap column in the same drop-to-drop manner to it, ensuring no airbubbles are introduced.
  10. End the run.

    11. Equilibriation of the ÄKTA:Start
  11. Move inlet tubes A and B from the ethanol flask to the MilliQ water flask.
  12. Initiate a manual run with a flow rate of 2ml/min in the software.
  13. Wash all tubing with 10CV of MilliQ by changing the flow path to include all waste tubes as well as the sample inlet and coupling tubes.
  14. End the run once all tubing has been washed.
  15. Move the inlet tubings A and B into the wash buffer and elution buffer respectively.
  16. Repeat steps 14-16 with the wash buffer only.

    17. Set-up before starting method
  17. Create a purification method in UNICORN
  18. Insert the sample inlet tube into the clarified lysate.
  19. Move the fractionation tubing and insert it into the holder on the Frac30 system.
  20. Insert sufficient eppendorf tubes into the Frac30 system.

    21. Running the method
  21. Run the method keeping a close eye on the system readings (UV, Conductivity, Pressure).
  22. Once the method has run, save the chromatogram and equilibriate the entire system as in steps 14-16 with MilliQ water, then with 70% ethanol.
  23. Buffer exchange can be done on the ÄKTA system as well, though the volume is limited to 1.5ml of sample. Therefore it is recommended to do a buffer exchange using PD10 Columns instead.

To seperate proteins by size via electrophoresis, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) was used. Molecular weight standard (SM) was applied in parallel to the samples to determine the protein size of the unknown sample.

Materials
  • Protein sample
  • Precast SDS-PAGE gel
  • PageRuler Prestained Protein Ladder
  • 3X sample loading buffer
  • 1X SDS running buffer (1x MES)
  • Gelcode Blue Safe Stain
Procedure

    Running the gel
  1. Add 3X sample loading buffer in a ratio of 1:3 to the protein sample (typically load around 2 μg of protein per sample). The final volume is dependent on the well size of the gel.
  2. Incubate samples for 5 minutes in a heating block at 95°C.
  3. Centrifuge samples at 13,000 rpm for 60 seconds.
  4. Remove the tape at the bottom of the precast gel cassette and assemble the cassette into the SDS-PAGE chamber.
  5. Remove the comb from the gel.
  6. Add running buffer to the inner and outer chambers.
  7. Load the samples (volume depending on well size) and protein ladder (5 μL) into the wells.
  8. Run the gel at 200V for 25 to 35 minutes in a cold room at 4°C, until the dye front reaches the reference line.
  9. Remove cassette from the chamber and remove the gel from the cassette using opening levers. Place the gel directly into a staining container with dH2O (see below).

    10. Staining the gel
  10. Place the gel in a staining container and add dH2O.
  11. Incubate for 3x 5 min on an orbital shaker, to wash the gel. Exchange dH2O in between the incubations.
  12. Remove the dH2O and add Gelcode Blue Safe Stain solution until it covers the gel.
  13. Incubate for 1 hour on an orbital shaker.
  14. Destain the gel in dH2O on an orbital shaker over night.
  15. Next day, take a picture of your gel using the imager.

For use on the gel obtained by SDS-PAGE.

Materials
  • Prepackaged turbo transblot PDVF membrane
  • Blocking buffer (PBS buffer with 5% milk poweder and 0.5% v/v Tween 20)
  • PBS-Tween (PBS buffer with 0.1% v/v Tween 20)
  • Anti-His-tag Antibody HRP conjugated
Procedure
  1. Note: to be started directly after removinf gel from the chamber.
  2. Transfer gel to a turbo transblot PDVF membrane with prepackaged westernblot membrane for tranturbo system
  3. Transfer of proteins from gel to membrane will be carried out via electroblotting for 10 min at 1.3A and 25V
  4. Post transfer take the membrane and put it in a 50 mL falcon tube.
  5. Add 5 ml blocking buffer to your tube.
  6. Place the tube on a rocking table for 1h.
  7. Wash the membrane by filling the tube with 20 mL PBS-Tween and put it on a rocking table for 5 min. Empty the tube of buffer.
  8. Repeat the washing two times (a total of three washes).
  9. Add Anti-His-tag Antibody HRP conjugate diluted 1:2500 in 5ml blocking buffer to the membrane. Incubate on a rocking table for 1 h.
  10. Wash the membrane as described above 3 times.
  11. Put the membrane in a plastic box with PBS-Tween for 5 min.
  12. Pour out the buffer.
  13. Dip the membrane in HRP substrate.
  14. Put the membrane in a plastic folder; mark the bands on the protein marker on the plastic film with a marker pen.
  15. Run the camera for blot development.

Protocol for analysis of NLuc-anchor peptide construct binding to 96-well plates. Different plate colours were used due to accessibility reasons.

Materials
  • Greiner Bio-One Polystyrene 96-well F-Bottom Microplates (White)
  • Greiner Bio-One Polypropylene 96-well F-Bottom Microplates (Black)
  • CLARIOstar Plus Microplate Reader
  • Promega NanoGlo Substrate
  • Purified, buffer exchanged NLuc-anchor peptide construct
  • 1x PBS
Procedure
  1. Make a dilution series using the NLuc construct to get 7 desired concentrations:
  2. 16µg/µl, 8µg/µl, 4µg/µl, 2µg/µl, 1µg/µl, 0.5µg/µl, and 0.25µg/µl were used in our testing.
  3. Mix the NanoGlo buffer with the NanoGlo substrate at a 50:1 ratio:
  4. Prepare 2x the volume required and an extra 1ml to ensure enough substrate for subsequent steps.
  5. Add 50µl of 1x PBS into the blank wells
  6. Add 50µl of the protein sample to the bottom of the sample wells.
  7. Add a subsequent 50µl of the NanoGlo substrate mix into the bottom of all the used wells (both blank and sample).
  8. Incubate the plates at room temperature without shaking for 5 minutes.
  9. Measure the luminescence using the plate reader and save the data for analysis.
  10. Discard all the liquid in the wells by quickly and decisively invert the well plate, over a sink/waste container, with force.
  11. Wash the used wells with 200µl of 1x PBS and wait 10-30 seconds and once again emptying the plate as in step 7.
  12. Repeat steps 7-8 2 more times.
  13. Add 50µl of 1x PBS into the bottom of all the used wells.
  14. Add a subsequent 50µl of the NanoGlo substrate mix into the bottom of all the used wells.
  15. Measure the Luminescense using the plate reader and save the data for analysis.