Experiments

In order to evaluate if our proteins would bind correctly to a chitin polymer, we performed a molecular docking analysis. We retrived a structure for the chitin octamer in PubChem (https://pubchem.ncbi.nlm.nih.gov/compound/Chitin-Octamer). For the chitinases, we searched their entry in the UniProt databank and retrived their structure available from AlphaFold: A0A179G878 for Pcchi44 and G3Y2N5 for CfcI. With the structures in hand, we performed the molecular docking simulation with SwissDock[1]. After retrieving the docked structures, we calculated the binding free energy with PRODIGY[2].

[1] GROSDIDIER, Aurelien; ZOETE, Vincent; MICHIELIN, Olivier. SwissDock, a protein-small molecule docking web service based on EADock DSS. Nucleic acids research, v. 39, n. suppl_2, p. W270-W277, 2011.

[2] XUE, Li C. et al. PRODIGY: a web server for predicting the binding affinity of protein–protein complexes. Bioinformatics, v. 32, n. 23, p. 3676-3678, 2016.

Materials

• Ice bath
• Incubator
• Aliquot of chemically competent E. coli bacteria
• Plasmid
• Liquid LB medium
• Solid LB medium with antibiotic
• Pipettes and tips
• Shaker
• Water bath
• 1,5ml microtubes
• Laminar flow hood

Methodology

1. Remove the bacteria from the -80ºC freezer and thaw on ice;
2. Turn on the water bath at 42ºC;
3. Thaw DNA plasmid on ice;
4. Add 1ul of DNA at the desired concentration to the tube containing the chemically competent bacteria;
5. Incubate the tube on ice for 30 min;
6. Heat shock in the bath at 42ºC for 30s;
7. Incubate immediately on ice for 2 min;
8. Add to the tube 600ul of LB liquid medium gently;
9. Incubate the tube at 37ºC under agitation in the shaker (~120rpm) for 1h30min;
10. Remove an aliquot (suggested: 200ul) and plate (Solid LB + antibiotic);
11. Incubate the plates with the bacteria overnight in the incubator at 37ºC;
12. Wait 16h and see if the bacteria have grown.

Materials

• Eppendorf tubes
• Falcon
• Ice bath
• Aliquot of E. coli bacteria
• Pipettes and tips
• Shaker
• Solutions: a)100mM CaCl2; b)100mM CaCl2 with 20% glycerol; LB;
• Laminar flow hood

Methodology

Day 01

1. Inoculate 50ul of the desired bacteria in 5ml of medium LB in a Falcon;
2. Leave the falcon overnight in the shaker at 37ºC;

Day 02

1. Transfer 1ml of pre-inoculum to 50ml of LB into an erlen;
2. Grow cells up to 5x10^7 (OD 0.4 - 0.5). Usually it takes around 1-2 hrs;
3. Incubate on ice for 10 min. From now on, everything must be done on ice;
4. Heat shock in the bath at 42ºC for 30s;
5. Incubate immediately on ice for 2 min;
6. Centrifuge at 4ºC, 4,000g for 8 min;
7. Discard the supernatant;
8. Resuspend in 25ml (1/2 of LB volume) with 100mM CaCl2;
9. Leave on ice for 5 min;
10. Centrifuge at 4ºC, 4,000g for 8 min;
11. Discard the supernatant;
12. Resuspend in 2ml (1/25 of LB volume) with 100mM of CaCl2+Glycerol;
13. Keep in refrigerator 4ºC for 12-24hrs;

Day 03

1. Dispense 50ul-200ul in Eppendorf tubes;
2. Store at -80ºC immediately;

Methodology

The tailing procedure consists of adding a single adenine overhang on the 3’ ends, to make the gene fragment compatible with TA cloning.

1. Add the following reagents to a 20uL reaction:
• Gene fragment: 50ng
• Taq polymerase: 1-3 units
• 10x Taq polymerase buffer (-Mg): 2ul
• 1mM dATP: to [0.2mM]
• 25mM MgCl2: to [1.5mM]
• Nuclease-free water: to final 20ul volume
2. Incubate at 70ºC for 30 minutes;

If necessary, it is possible to add more quantity of Gene fragment. After the shutdown of the freezer we decided to add more gene fragment because we were scared our DNA could have been degraded. This protocol was taken from the document: “TA cloning method” from IDT[1].

References

[1]gBlocks and gBlocks HiFi Gene Fragments | IDT. (n.d.). Integrated DNA Technologies. Retrieved September 21, 2022, from https://www.idtdna.com/pages/products/genes-and-gene-fragments/double-stranded-dna-fragments/gblocks-gene-fragments

Materials

• Petri dishes containing culture medium
• Sterile handle
• Incubator
• Laminar flow hood

Methodology

Note: Perform every step inside a laminar hood flow.

1. Remove a microbial aliquot from the anterior tube or plate with the sterile handle;
2. Open the new plate;
3. Touch the solid medium of the new plate with the sterile handle;
4. After cultivation is complete, close the plate and identify it;
5. Incubate at room temperature with the lid facing down;

Materials

• Autoclaved drinking straw
• Autoclaved toothpick
• Liquid medium
• Autoclaved bottles of 250ml
• Fungus in solid medium
• Shaker

Methodology

Note: Perform every step inside a laminar hood flow.

1. Open the autoclaved bottle;
2. Transfer 100ml of liquid medium to the bottle;
3. Open the fungus in solid medium and with the straw make 10 holes in the agar. As a result there will be 10 fungi plugs for each bottle;
4. Pick up the 10 plugs with the toothpick and transfer them to the bottle with the liquid medium;
5. Close the bottle and transfer it for growth in the Shaker, at a rotation of 150rpm. The bottle should be half open in the Shaker for aeration;

Methodology

Bacteria preparation

1. Grow bacteria on MRS agar overnight;
2. Suspend colonies in 5 mL of 0.85% NaCl solution until reaching the turbidity of 1.0 McFarland (3.0 x 10^8 CFU/mL) or up to OD600 = 0.5 in 0.9% NaCl;
3. Dilute inoculum 1:1000 in MRS broth to inoculate on a microplate;

Antibiotic preparation

1. Weight the proper amount of antibiotic in a microtube;
2. Add sterile MiliQ water or ethanol in a laminar flow cabinet and vortex;
3. Filter solution to a sterile microtube with a 0.2 μm filter;
4. Store at freezer or fridge, according to manufacturer’s instructions;

Plate setting up

1. Add 100 uL of MRS broth into each well (except into first column);
2. Add double volume of antibiotic into first column and make serial dilutions, withdrawing 100 uL from each well and adding into the next (mix by pipetting). Discard the withdrawn solution from last column;
3. Add 100 uL of bacterial suspension into each well;
4. Incubate the plate in anaerobic conditions at 37 °C for 48 h. Select minimal antibiotic concentration the inhibits bacterial growth;

References:

Dec, Marta, et al. “Identification and antibiotic susceptibility of lactobacilli isolated from turkeys”. BMC Microbiology, vol. 18, no 1, outubro de 2018, p. 168. BioMed Central, https://doi.org/10.1186/s12866-018-1269-6.

“Antibiotic Susceptibility of Members of the Lactobacillus Acidophilus Group Using Broth Microdilution and Molecular Identification of Their Resistance Determinants”. International Journal of Food Microbiology, vol. 144, no 1, novembro de 2010, p. 81–87.

Methodology

To extract the DNA from the agarose gel bands, we used NucleoSpin® Gel and PCR Clean-up kit [1]. We adapted the protocol from the kit.

Excise DNA fragment / solubilize gel slice

1. Excise the DNA fragment from the agarose gel with a scalpel;
2. Determine the weight of the gel slice and transfer it to a clean tube;
3. For each 100 mg of agarose gel < 2 % add 200 μL Buffer NTI;
4. Incubate sample for 10 min at 50 °C;

Bind DNA

1. Place a NucleoSpin® Gel and PCR Clean-up Column into a Collection Tube (2 mL) and load up to 700 μL sample;
2. Centrifuge for 30 s at 11000 g. Discard flow-through and place the column back into the collection tube. Load remaining sample if necessary and repeat the centrifugation step;

Wash silica membrane

1. Add 700 μL Buffer NT3 to the NucleoSpin® Gel and PCR Clean-up Column;
2. Centrifuge for 30 s at 11000 g;
3. Add 100 uL of bacterial suspension into each well;
4. Discard flow-through and place the column back into the collection tube;

Dry silica membrane

1. Centrifuge for 1 min at 11000 g to remove Buffer NT3 completely;

Elute DNA

1. Place the NucleoSpin® Gel and PCR Clean-up Column into a new 1.5 mL microcentrifuge tube;
2. Add 15μL Buffer NE and incubate at room temperature for 1 min. Centrifuge for 1 min at 11000 g;

References:

PCR clean-up Gel extraction User manual NucleoSpin ® Gel and PCR Clean-up Ordering information Ordering information. (2017). https://www.takarabio.com/documents/User%20Manual/NucleoSpin%20Gel%20and%20PCR%20Clean/NucleoSpin%20Gel%20and%20PCR%20Clean-up%20User%20Manual_Rev_04.pdf

Materials

• Solution I(10ml): Tris HCl 1M pH8.0 (0,25mL) + EDTA pH8.0 0,5M (0,2mL) + Glucose 500mM(1mL) + Distilled water(8,55mL);
• Solution II(10ml): Distilled water (8mL) + NaOH 2M (1mL) + SDS 10% (1mL);
• Solution III: Potassium acetate 5M pH 5.5 + Acetic acid (11,5 mL) + Distilled water (28,5 mL). Adjust the pH to 5.5;

Methodology

1. Inoculate 5 mL of LB broth (with antibiotic, if bacteria were transformed) a day before;
2. Centrifuge the inoculum in 2 mL microtubes for 2 min at 10000 RPM and discard supernatant;
3. Add 200 uL of Solution I + 20 uL of RNAse 10 mg/mL (10% v/v in relation to Solution I). Gently agitate to resuspend cells. Incubate at room temperature for 10 min;
4. Add 200 uL of freshly-prepared, cold Solution II. Shake tubes by invertion 6 times and incubate on ice for 5 min;
5. Centrifuge at 12000 RPM for 10 min;
6. Transfer about 400 uL of supernatant to clean tubes;
7. Add 800 uL of cold, pure ethanol or isopropanol and gently shake by inversion. Incubate at -20°C for 1.5-2 h. The ethanol/isopropanol volume add is twice the amount taken from the supernatant in the previous step;
8. Centrifuge at 14000 RPM for 15 min. Discard supernatant
9. Add 500 uL of cold ethanol 70% to pellet. Centrifuge at 14000 RPM for 15 min and discard supernatant;
10. Dry pellet at room temperature;
11. Resuspend in 30 uL of TE buffer or sterile MiliQ water;
12. Store at -20 °C;

Methodology

Agarose gel preparation (1%):

1. For 50ml of gel, weigh 0.5g of agarose;
2. Add 50ml of TAE 1X;
3. Melt the solution in the microwave until homogenized;
4. After the cooling of the gel solution, add 5ul of SYBR Safe stain[1]. Mix the solution;
5. Pour the gel solution into the holder without bubbles;
6. Place the comb of the desired thickness on the holder;
7. Wait for the solution to solidify;
8. Place the gel holder in the electrophoresis box. Add TAE buffer 1X until the gel surface is covered;

Note:Alternatively, the gel can also be wrapped in plastic wrap and stored at 4°C until use

Samples preparation:

1. Add Loading dye to the samples, achieving the concentration of 1X. Gel loading dye is typically made at 6X concentration;

Gel electrophoresis:

1. Carefully load the DNA samples into the gel wells. An appropriate DNA ladder, according to your DNA length, should always be loaded along with experimental samples. We use 5uL of ladder;
2. Program the power supply to desired voltage. We use 100V;
3. Attach the leads of the gel box to the power supply. Pay attention to the colors, red and black;
4. Turn on the power supply and verify if it is working: seek bubbles coming from the wires;
5. Run the gel until the dye has migrated to an appropriate distance. We usually stop the experiment when there is a distance of one thinger between the two colors;

Observation of the DNA bands:

1. When electrophoresis is completed, turn off the power supply and remove the lid of the gel box;
2. Remove gel from the gel box. Drain off excess buffer from the surface of the gel;
3. Expose the gel to UV light. DNA bands should show up as green fluorescent bands. Take a picture of the gel;



References:

[1]SYBR Safe - DNA Gel Stain - US. (n.d.). Www.thermofisher.com. Retrieved September 21, 2022, from https://www.thermofisher.com/br/en/home/life-science/dna-rna-purification-analysis/nucleic-acid-gel-electrophoresis/dna-stains/sybr-safe.html#:~:text=SYBR%20Safe%20stain%20is%20specifically

[2] Lee, P. Y., Costumbrado, J., Hsu, C.-Y., & Kim, Y. H. (2012). Agarose Gel Electrophoresis for the Separation of DNA Fragments. Journal of Visualized Experiments, 3923(62). https://doi.org/10.3791/3923

Materials

• Pipettes and tips;
• Microtubes;

Methodology

1. Add the following reagents from the kit to a tube to perform a 5ul reaction:
• A-tailed gBlocks: 0,5-3ul
• Salt Solution: 1ul
• Water: to complete 5ul
• TOPO vector: 1ul
2. Incubate in room temperature for 30 minutes;
3. Transform the result in cloning bacterial strains, plate with IPTG and Xgal (for Blue-White Screening) and Amp (Selection);

References:

[1]TOPO ® TA Cloning ® Kit. https://tools.thermofisher.com/content/sfs/manuals/topota_man.pdf

Methodology

We adapted this portocol from NEBcloner[1].

1. Add the following reagents to a tube for a 25ul reaction:
• DNA: 3-9ul
• 10X Buffer EcoRI: 3ul
• Water: to complete 25ul
• EcoRI: 1-3ul
2. Incubate at 37°C for 30 minutes;

References:

[1]NEBcloner. Nebcloner.neb.com. https://nebcloner.neb.com/#

Methodology

We adapted this portocol from Promega[1].

1. Add the following reagents to a tube for a 10ul reaction:
• DNA (1ug/ul): 7,9ul
• Buffer H: 1ul
• Acetyled BSA (10ug/ul): 0,1ul
• PstI (10U/ul): 1ul
2. Incubate at 37°C for 3 hours;

References:

[1]Promega. Assembly of Restriction Enzyme Digestions. 2011.

Methodology

We adapted the protocol from Puregene® Concentrating DNA protocol (Qiagen, Germany) described by the manufacturer.

1. Add 0.5 volumes of Protein Precipitation Solution (Qiagen, Germany) and 2 volumes of ethanol to digested product;
2. Incubate at room temperature for 15 min;
3. Centrifuge at 13000 rpm for 5 min;
4. Discard the supernatant;
5. Add 3 volumes of 70% ethanol to the tube;
6. Centrifuge at 13000 rpm for 1 min;
7. Discard the supernatant;
8. Let the samples dry with the tubes upside down and open;

References:

[1]Puregene® DNA Handbook.

Methodology

We used the protocol from Promega PureYield™ kit.

Prepare Lysate

1. Add 600 µL of bacterial culture to a 1.5ml microcentrifuge tube;
2. Add 100 µL of Cell Lysis Buffer, and mix by inverting the tube 6 times;
3. Add 350 µL of cold (4–8°C) Neutralization Solution, and mix thoroughly by inverting;
4. Centrifuge at maximum speed in a microcentrifuge for 3 minutes;
5. Transfer the supernatant (~900 µL) to a PureYield™ Minicolumn without disturbing the cell debris pellet;
6. Place the minicolumn into a Collection Tube, and centrifuge at maximum speed in a microcentrifuge for 15 seconds;
7. Discard the flowthrough, and place the minicolumn into the same Collection Tube;

Wash

1. Add 200 µL of Endotoxin Removal Wash (ERB) to the minicolumn;
2. Centrifuge at maximum speed in a microcentrifuge for 15 seconds;
3. Add 400 µL of Column Wash Solution (CWC) to the minicolumn;
4. Centrifuge at maximum speed in a microcentrifuge for 30 seconds;

Elute

5. Transfer the minicolumn to a clean 1.5 mL microcentrifuge tube, then add 30 µL of Elution Buffer or nuclease-free water directly to the minicolumn matrix;
6. Incubate for 1 minute at room temperature;
7. Centrifuge for 15 seconds to elute the plasmid DNA;
8. Cap the microtube, and store eluted plasmid DNA at –20°C;

References:

[1]Promega PureYield™ kit.

Methodology

Preparation of electrocompetent cells

1. Cultivate Lactobacillus in MRS. Incubate at 37°C, overnight, without shaking. Preferably in an anaerobiosis jar;
2. Dilute pre-culture with 100 mL of MRS-glycine 1%;
3. Grow the cells at 37 °C, without shaking, until reaching OD = 0.6-0.7 (about 5 hours);
4. When reaching the specific OD, keep the tubes always on ice for the next steps;
5. Transfer the 100 mL to 4 falcons with 25 mL each;
6. Centrifuge tubes for 15 min, at 5500 rpm and 4 °C;
7. Wash the pellet 4 times with 10 mL of ice cold MgCl2 10 mM, centrifuging the suspension for 10 minutes at 5000 rpm after every wash;
8. Resuspend the pellet with 5 mL of ice cold sucrose 0.5 M + glycine 10% and divide the material between two falcons;
9. Wash pellet twice with 20 mL of ice cold sucrose 0.5 M + glycine 10% solution;
10. Centrifuge for 15 min at 5000 RPM, 4 °C after every wash;
11. Resuspend the pellet with 5 mL of ice cold sucrose 0.5 M + glycine 10% and divide the material between two falcons;
12. Wash the pellet once with 20 mL of ice cold sucrose 0.5 M + glycine 10% solution;
13. Centrifuge for 15 min at 5000 RPM, 4 °C;
14. Resuspend the pellet gently in 1 mL of cold PEG 3000 30% + glycerol 10%;
15. Store aliquots of cold cells at -80 °C;

Electroporation

1. Thaw 100 uL electrocompetent cells from -80 °C on ice;
2. Keep all tubes on ice before use;
3. Mix 1 ug of DNA with the competent cells and place tubes on ice for 10 min;
4. Transfer cells from tubes to a cold electroporation cuvette (2.0mm);
5. Electroporar: 2500V, 25 uF y 200 Ω;
6. Immediately add 900 uL of MRS supplemented with sucrose 500 mM and MgCl2 80 mM to the cuvette;
7. Transfer cells to a microtube and incubate tubes for 3h at 37 °C;
8. Plate 100 uL of culture on MRS agar with the resistance encoded antibiotics at 37 °C for 24-48h in anaerobic conditions;

Methodology

Note: It is recommended to perform a negative control without template DNA to verify if the primers are contaminated

1. Add the following reagents in a tube;
• Buffer without Mg: 1X
• DNTP Mix: 0.2mM
• MgCl2: 1.5mM
• Taq polymerase: 1-2.5 Units
• Forward primer: 0.5uM
• Reverse primer:0.5uM
• Template DNA(200pg/ul): 2ul
• Water: to complete 50uL
2. Put the tube inside the thermocycler and close the thermocycler;
3. Adjust the thermocycler:
• Initial denaturation: 94°C 3min
• Denaturation: 94°C 45s
• Annealing: 54°C 30s
• Extension: 72°C 2min 15s (90s per Kb)
• Final extension: 72°C 10min
• Wait: 4°C infinite
4. Perform gel electrophoresis to verify if your sequence of interest was amplified;

Materials

• Solution A: 29.2g acrylamide page, 0.8g Bis, complete to 100ml distilled water
• Solution B: 18.15g Tris, pH8.8, complete to 100ml distilled water
• Solution C: 6g Tris, complete to 100ml distilled water
• Solution SDS 10%
• Solution PSA 10%
• Running Buffer 1X: 25mM Tris, 0.1% SDS, 0.25M Glicine, Water
• Decoloring solution: 40% ethanol, 10% acetic acid
• Comassie Blue dye
• TEMED
• TA 4X Buffer

Methodology

Preparation of the separation gel (12%)

1. Add 4ml of Solution A in a falcon tube;
2. Add 2.5ml of Solution B;
3. Add 0.1ml of SDS 10%;
4. Add 3.35ml of distilled water;
5. Add 70uL of PSA 10%;
6. Add 7uL of TEMED;
7. Mix the solution in the falcon;
8. Add the solution inside the glass gel support. Wait 30 min for the separation gel to polymerize;

Preparation of the concentration gel

1. Add 325uL of Solution A in a falcon tube;
2. Add 625uL of Solution C;
3. Add 25uL of SDS 10%;
4. Add 1.525ml of distilled water;
5. Add 35uL of PSA 10%;
6. Add 7uL of TEMED;
7. Mix the solution in the falcon;
8. Add the solution inside the glass gel support, on top of the separation gel. Put the desired comb in the gel. Wait 30 min for the concentration gel to polymerize;

Preparation of the samples

1. Mix 15ul of your sample with 5ul TA 4X Buffer;

Assembly of the electrophoresis

1. Put the glass gel support vertically inside the electrophoresis box. Fill the box with Running Buffer 1X until the gel is covered;
2. Add the prepared samples in the gel wells. Do not forget to add an appropriate protein molecular weight ladder in the first well;
3. Assemble the running unit. Attach the leads of the gel box to the power supply. Define the voltage of 70V for 30min and then change for 150V for 1h;

Coloring the gel with Comassie Blue

1. Put the gel in a plastic container and add Comassie Blue dye. Incubate for 1 hour at room temperature with gentle shaking;
2. Wash the gel with 100 mL of Decoloring solution for 1–3 hours with gentle shaking. If needed, change the decoloring solution from time to time;
3. Observe the gel with a white surface below it. Take pictures;

Methodology

1. Treat 20g of sieved crab shell flakes chitin with 150 mL of ~12M concentrated HCl (Fuming hydrochloric acid 37%);
2. Add the HCl slowly with continuous stirring with a glass pipette for 5 minutes;
3. Continue to stir for 1 minute every 5 minutes during an hour in a chemical fume hood at room temperature (25°C);
4. Pass the chitin-HCl mixture through 8 layers of cheesecloth to remove large chitin chunks (about 100 mL obtained);
5. Treat the filtrate with 2 liters of ice cold distilled water to allow precipitation of colloidal chitin and incubate it overnight at 4°C;
6. Pass the solution through two layers of coffee filter paper housed in a Buchner funnel seated in a vacuum filtration flask under vacuum;
7. Add approximately 3 liters of tap water (pH of ~8.0) through the colloidal chitin using the filter assembly, until the pH of the filtrate has risen to 7.0 (estimated by pH paper);
8. Press the colloidal chitin between the coffee filter papers to remove additional moisture and then place it in a 100 mL beaker covered with two layers of aluminum foil;
9. Sterilize it by autoclaving at standard temperature and pressure and store it at 4°C until further use;
10. To prepare chitin agar plates, add 0.2% dry powdered colloidal chitin or 2.0% moist colloidal chitin to the other components;

References:

N. Murthy and B. Bleakley, "Simplified Method of Preparing Colloidal Chitin Used For Screening of Chitinase- Producing Microorganisms", The Internet Journal of Microbiology, vol. 10, issue2, pp.7, 2012.

Methodology

We adapted this protocol from NEB. Before initiating the protocol, it is necessary to verify the amount of DNA that needs to be added by calculating the wanted molar ratio (We recommend 1:3 vector:insert)

1. Add 2ul of T4 DNA Ligase Buffer 10x in a microtube;
2. Add 50ng of the DNA vector;
3. Add 37.5 ng of the DNA insert;
4. Complete the reaction volume (20ul) with Nuclease-free water;
5. Add 1ul of T4 DNA ligase (5U);
6. Mix the reaction by pipetting up and down;
7. Incubate the reaction in the thermocycler at 16ºC overnight;

References:

Ligation Protocol with T4 DNA Ligase (M0202), NEB.

Materials

• 20% ethanol
• His GraviTrap column;
• Distilled Water;
• Stripping buffer: 20 mM sodium phosphate, 500 mM NaCl, 50 mM EDTA, pH 7.4;
• Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4;
• Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4;

Note: Pass the buffer solutions through a 0.22 µm or a 0.45 µm filter before applying them to the column

Methodology

We adapted this protocol from the Affinity Chromatography Handbook (Cytiva). We used a 2ml His GraviTrap column to purify His-tagged proteins.

Stripping, recharging and cleaning the column

1. Strip the chromatography medium by washing with at least 5 to 10 column volumes of stripping buffer;
2. Wash with at least 5 to 10 column volumes of binding buffer;
3. Immediately wash with 5 to 10 column volumes of distilled water;
4. Wash the column with 1M NaOH, contact time usually 1 to 2 h (12 h or more for endotoxin removal).;
5. Immediately wash with approximately 10 column volumes of binding buffer, followed by 5 to 10 column volumes of distilled water. (5U);
6. Recharge the water-washed column by loading 0.5 column volumes of 0.1M NiSO4 in distilled water onto the column;
7. Wash with 5 column volumes of distilled water, and 5 column volumes of binding buffer (to adjust pH) before storage in 20% ethanol;

Blank run

1. Wash the column with 5 column volumes of distilled water (to remove the 20% ethanol);
2. Wash with 5 column volumes of elution buffer, and collect the eluate;
3. Equilibrate with 10 column volumes of binding buffer;

Sample preparation

1. Dilute the sample to a final result of 50% binding buffer;
2. Add 0.5 to 35 mL of the prepared sample. (5U);
3. Pass the sample through a 0.22 µm or a 0.45 µm filter before applying it to the column;

Protein purification

1. Equilibrate the column with 10 mL of binding buffer;
2. Add 0.5 to 35 mL of the prepared sample. (5U);
3. Wash with 10 mL of binding buffer;
4. Apply 3 mL of elution buffer and collect the eluate. Under denaturing conditions, elute twice with 3 mL of elution buffer;

References:

Vol. 2: Tagged proteins, Affinity Chromatography Handbook, Cytiva.