Goal: To create a functional and nutritional liquid or solid medium for bacteria growing. Activation, cultivation, and fermentation for microorganisms.
Materials:
1) Conical flask
2) Autoclave
3) Tryptone
4) Yeast Extract
5) Sodium Chloride
6) Double distilled H2O
7) Agar
Procedures:
1) Add 10 g tryptone, 5 g yeast extract, and 10 g of sodium chloride per liter of the culture medium. (Note: 1.5% agar powder was added to the solid culture medium);
2) Add into 1L conical flask;
3) Maintain at 121 ℃ for 20 minutes in an autoclave for sterilization.
Experiments
1. Preparation of LB (Luria-Bertani) culture medium
2. Construction of recombination plasmids
pET-28a(+)-NF-L/Aβ-42/α-syn and pET-28a(+)-NF-L/Aβ-42/α-syn-mcherry
Ⅰ. Obtain target gene fragments by PCR
Goal: To amplify target genes fragments for Infusion recombination.
Materials:
1) 2x PrimeStarMix
2) Gene template
3) Double distilled water (ddH2O)
4) Primer-F
5) Primer-R
6) PCR tube
7) PCR thermal cycler
* PrimeStarMix includes buffer and dNTPs for PCR. The extention rate for PrimeStarMix (2x) is 1000bp/10 s.
Procedures:
1) Add 2x PrimerStarMix 25µl, gene template 1µl, primer-F 1µl, and primer-R 1µl to centrifuge tube. Add ddH2O to the centrifuge tube up to 50µl. If bubbles are observed in the mixture, centrifuge the mixture for a few seconds to remove the bubbles;
2) Insert PCR tubes into PCR thermal cycler. Pre-denature the fragments at 95˚C for 3 minutes;
3) Perform 35 cycles of 30 seconds 95˚C denaturing, 30 seconds 55˚C annealing, and 2 minutes extension 72˚C extension;
4) Extend the fragments for 1 minutes at 72˚C;
5) If not use immediately, preserve the fragments at 4˚C;
6) Recover PCR fragments by agarose gel electrophoresis.
Ⅱ. Linearize pET-28a(+) backbone by restriction endonucleases
Goal: To linearize pET-28a(+) backbone for Infusion recombination.
Materials:
1) pET-28a(+) backbone plasmid
2) 10x Cutting buffer
3) EcoRI endonuclease
4) XhoI endonuclease
5) Double distilled water (ddH2O)
Procedures:
1) Add 5µg backbone plasmid, 10x Cutting buffer 5µl, EcoRI endonuclease 5µl, and XhoI endonuclease 5µl in PCR tube. Add ddH2O to PCR tube up to 50µl;
2) Mix by pipette and remove the bubbles by low-speed centrifuge;
3) Insert PCR tubes into PCR thermal cycler. Digest the plasmid at 37˚C for 30 minutes;
4) Recover linearized pET-28a(+) backbone fragments by agarose gel electrophoresis.
Ⅲ. Agarose gel construction, deployment and recycle
Goal: To produce a functional and well-shaped gel for electrophoresis.
Materials:
1) Casting Tray
2) Well comb
3) Microwave
4) 1x TAE buffer
5) Agarose M
6) 10000x Nucleic acid gel stain
7) Conical flask
Procedures:
1) Dissolve 1.5g Agarose in 200mL Conical flask;
2) Add 100mL TAE buffer;
3) Heat the material with microwave;
4) Melt the mixture at 30 sec interval;
5) See whether bubbles are forming, if did succesfully mixed;
6) Add 3μl nucleic acid gel stain (10000x);
7) Let it cool for 5~10 minutes;
8) Pour the agarose mixture into the casting tray;
9) Place the appropriate well comb to create the wells;
10) Wait to solidify ~30 minutes;
11) Remove the comb and place the gel in the gel box.
Ⅳ. Agarose gel electrophoresis
Goal: To verify the DNA bands' lengths and thus preparing for the next step of gel extraction to recover the bands that are of the correct length.
Procedures:
1) Extract 50µl of every sample; add 5µl of loading buffer (10x) to each sample;
2) Place the gel in the horizontal electrophoresis system;
3) Extract 50µl of the mixed solution containing the sample, mix them till their colors are almost uniform;
4) Add the marker to the first well and load 50µl of each sample into the wells;
5) The procedure runs at 120V for 20min;
6) Take out the gel from the device and proceed to following steps.
Ⅴ. DNA gel extraction
Goal: To recover DNA fragments in agarose gel.
Materials:
1) Agarose gel with target genes fragments and linearized pET-28a(+) backbone;
2) 1.5 mL Eppendorf (EP) tube
3) Agarose Gel Electrophoresis Recovery Kit
Procedures:
1) Cut the slice of gel containing target genes fragments and linearized pET-28a(+) backbone, cutting off as much unneeded gel as possible, and place it in an 1.5 mL EP tube;
2) Add 500 microliters of buffer B2 and put the tube into a 50°C hot water bath until gel has completely melted;
3) Transfer the solution containing melted gel into a purification column that's in a collection tube and perform centrifugation at 12000 rpm for 30 seconds;
4) Empty the collection tube, put the purification column back in, and add 500 microliters of wash solution containing pure ethanol. Perform centrifugation at 12000 rpm for 30 seconds, and empty collection tube again;
5) Repeat steps 4;
6) Perform centrifugation one more time at 12000 rpm for 2 minutes, then open the cap of the tube and let it sit for one minute to allow the ethanol to evaporate;
7) Transfer purification column to a new 1.5 mL EP tube. Add 50 microliters of double distilled water (ddH2O) at the center of the purification column;
8) Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 12000 rpm for 1 minute;
9) Discard the purification column. Store DNA fragments in the EP tube at 4°C.
Ⅵ. Homologous recombination of linearized pET-28a(+) backbone with target gene fragments
Goal: Perform homologous recombination between the linearized backbone and target gene fragments to generate the complete plasmid.
Materials:
1) CloneExpress Mix (2x) br 2) PCR products of NF-L, Aβ-42, α-syn, and mcherry
3) Linearized pET-28a(+) backbone
Procedure:
1) Measure the concentration of the gel-recovered fragments;
2) Prepare the system for the construction of pET-28a(+)-NF-L/Aβ-42/α-syn in PCR tubes: 5μl CloneExpress Mix, 2.5μl Linearized pET-28a(+) backbone, and 2.5μl NF-L or Aβ-42 or α-syn fragments;
3) Prepare the system for the construction of pET-28a(+)-NF-L/Aβ-42/α-syn-mcherry in PCR tubes: 5μl CloneExpress Mix, 2μl Linearized pET-28a(+) backbone, 1.5μl NF-L or Aβ-42 or α-syn fragments, and 1.5μl mcherry fragment;
4) Insert PCR tubes into PCR thermal cycler. Ligase the fragments at 50˚C for 30 minutes;
Ⅶ. Transformation
Materials:
1) Ligated recombination plasmid
2) Liquid LB solution without antibiotics
3) LB Agar plates with Kanamycin antibiotics
Procedures:
1) Add recombination plasmid into E.Coli BL21 strain;
2) Placed on ice for 20 minutes;
3) Heat shock at 42˚C for 45 seconds, then immediately put on ice for 2-3 minutes;
4) Add 400μL liquid LB solution without antibiotics, incubate at 37 ˚C for 30 minutes;
5) Centrifuge at 5000 rpm for 3 minutes, extract 300μL of supernatant then discard;
6) Apply bacterial liquid to LB Agar plates with Kanamycin antibiotics, incubate for 12-16 hours at 37˚C.
Ⅷ. Colony PCR verification
Goal: E.Coli BL21 strain containing constructed plasmids has multiplied. We aimed to identify mono-colony E.coli BL21 containing correct recombination plasmids by colony PCR.
Materials:
1) Taq Master Mix (2x)
2) Double distilled water (ddH2O)
3) Culture plates post-transformation
Procedures:
1) Add 10μl Taq Master Mix, 1μl primer-F, 1μl primer-R, and 8μl ddH2O into PCR tubes;
2) Pick a single colony with a 10μl sterile pipette tip and perform aspiration and dispensing in PCR tubes;
3) Insert mixture into a PCR thermal cycler, design the PCR program according to the manufacturer's instructions.
4) Perform agarose gel electrophoresis, and the presence of the correct bands indicates the successful construction of recombination plasmids.
Ⅰ. Obtain target gene fragments by PCR
Goal: To amplify target genes fragments for Infusion recombination.
Materials:
1) 2x PrimeStarMix
2) Gene template
3) Double distilled water (ddH2O)
4) Primer-F
5) Primer-R
6) PCR tube
7) PCR thermal cycler
* PrimeStarMix includes buffer and dNTPs for PCR. The extention rate for PrimeStarMix (2x) is 1000bp/10 s.
Procedures:
1) Add 2x PrimerStarMix 25µl, gene template 1µl, primer-F 1µl, and primer-R 1µl to centrifuge tube. Add ddH2O to the centrifuge tube up to 50µl. If bubbles are observed in the mixture, centrifuge the mixture for a few seconds to remove the bubbles;
2) Insert PCR tubes into PCR thermal cycler. Pre-denature the fragments at 95˚C for 3 minutes;
3) Perform 35 cycles of 30 seconds 95˚C denaturing, 30 seconds 55˚C annealing, and 2 minutes extension 72˚C extension;
4) Extend the fragments for 1 minutes at 72˚C;
5) If not use immediately, preserve the fragments at 4˚C;
6) Recover PCR fragments by agarose gel electrophoresis.
Ⅱ. Linearize pET-28a(+) backbone by restriction endonucleases
Goal: To linearize pET-28a(+) backbone for Infusion recombination.
Materials:
1) pET-28a(+) backbone plasmid
2) 10x Cutting buffer
3) EcoRI endonuclease
4) XhoI endonuclease
5) Double distilled water (ddH2O)
Procedures:
1) Add 5µg backbone plasmid, 10x Cutting buffer 5µl, EcoRI endonuclease 5µl, and XhoI endonuclease 5µl in PCR tube. Add ddH2O to PCR tube up to 50µl;
2) Mix by pipette and remove the bubbles by low-speed centrifuge;
3) Insert PCR tubes into PCR thermal cycler. Digest the plasmid at 37˚C for 30 minutes;
4) Recover linearized pET-28a(+) backbone fragments by agarose gel electrophoresis.
Ⅲ. Agarose gel construction, deployment and recycle
Goal: To produce a functional and well-shaped gel for electrophoresis.
Materials:
1) Casting Tray
2) Well comb
3) Microwave
4) 1x TAE buffer
5) Agarose M
6) 10000x Nucleic acid gel stain
7) Conical flask
Procedures:
1) Dissolve 1.5g Agarose in 200mL Conical flask;
2) Add 100mL TAE buffer;
3) Heat the material with microwave;
4) Melt the mixture at 30 sec interval;
5) See whether bubbles are forming, if did succesfully mixed;
6) Add 3μl nucleic acid gel stain (10000x);
7) Let it cool for 5~10 minutes;
8) Pour the agarose mixture into the casting tray;
9) Place the appropriate well comb to create the wells;
10) Wait to solidify ~30 minutes;
11) Remove the comb and place the gel in the gel box.
Ⅳ. Agarose gel electrophoresis
Goal: To verify the DNA bands' lengths and thus preparing for the next step of gel extraction to recover the bands that are of the correct length.
Procedures:
1) Extract 50µl of every sample; add 5µl of loading buffer (10x) to each sample;
2) Place the gel in the horizontal electrophoresis system;
3) Extract 50µl of the mixed solution containing the sample, mix them till their colors are almost uniform;
4) Add the marker to the first well and load 50µl of each sample into the wells;
5) The procedure runs at 120V for 20min;
6) Take out the gel from the device and proceed to following steps.
Ⅴ. DNA gel extraction
Goal: To recover DNA fragments in agarose gel.
Materials:
1) Agarose gel with target genes fragments and linearized pET-28a(+) backbone;
2) 1.5 mL Eppendorf (EP) tube
3) Agarose Gel Electrophoresis Recovery Kit
Procedures:
1) Cut the slice of gel containing target genes fragments and linearized pET-28a(+) backbone, cutting off as much unneeded gel as possible, and place it in an 1.5 mL EP tube;
2) Add 500 microliters of buffer B2 and put the tube into a 50°C hot water bath until gel has completely melted;
3) Transfer the solution containing melted gel into a purification column that's in a collection tube and perform centrifugation at 12000 rpm for 30 seconds;
4) Empty the collection tube, put the purification column back in, and add 500 microliters of wash solution containing pure ethanol. Perform centrifugation at 12000 rpm for 30 seconds, and empty collection tube again;
5) Repeat steps 4;
6) Perform centrifugation one more time at 12000 rpm for 2 minutes, then open the cap of the tube and let it sit for one minute to allow the ethanol to evaporate;
7) Transfer purification column to a new 1.5 mL EP tube. Add 50 microliters of double distilled water (ddH2O) at the center of the purification column;
8) Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 12000 rpm for 1 minute;
9) Discard the purification column. Store DNA fragments in the EP tube at 4°C.
Ⅵ. Homologous recombination of linearized pET-28a(+) backbone with target gene fragments
Goal: Perform homologous recombination between the linearized backbone and target gene fragments to generate the complete plasmid.
Materials:
1) CloneExpress Mix (2x) br 2) PCR products of NF-L, Aβ-42, α-syn, and mcherry
3) Linearized pET-28a(+) backbone
Procedure:
1) Measure the concentration of the gel-recovered fragments;
2) Prepare the system for the construction of pET-28a(+)-NF-L/Aβ-42/α-syn in PCR tubes: 5μl CloneExpress Mix, 2.5μl Linearized pET-28a(+) backbone, and 2.5μl NF-L or Aβ-42 or α-syn fragments;
3) Prepare the system for the construction of pET-28a(+)-NF-L/Aβ-42/α-syn-mcherry in PCR tubes: 5μl CloneExpress Mix, 2μl Linearized pET-28a(+) backbone, 1.5μl NF-L or Aβ-42 or α-syn fragments, and 1.5μl mcherry fragment;
4) Insert PCR tubes into PCR thermal cycler. Ligase the fragments at 50˚C for 30 minutes;
Ⅶ. Transformation
Materials:
1) Ligated recombination plasmid
2) Liquid LB solution without antibiotics
3) LB Agar plates with Kanamycin antibiotics
Procedures:
1) Add recombination plasmid into E.Coli BL21 strain;
2) Placed on ice for 20 minutes;
3) Heat shock at 42˚C for 45 seconds, then immediately put on ice for 2-3 minutes;
4) Add 400μL liquid LB solution without antibiotics, incubate at 37 ˚C for 30 minutes;
5) Centrifuge at 5000 rpm for 3 minutes, extract 300μL of supernatant then discard;
6) Apply bacterial liquid to LB Agar plates with Kanamycin antibiotics, incubate for 12-16 hours at 37˚C.
Ⅷ. Colony PCR verification
Goal: E.Coli BL21 strain containing constructed plasmids has multiplied. We aimed to identify mono-colony E.coli BL21 containing correct recombination plasmids by colony PCR.
Materials:
1) Taq Master Mix (2x)
2) Double distilled water (ddH2O)
3) Culture plates post-transformation
Procedures:
1) Add 10μl Taq Master Mix, 1μl primer-F, 1μl primer-R, and 8μl ddH2O into PCR tubes;
2) Pick a single colony with a 10μl sterile pipette tip and perform aspiration and dispensing in PCR tubes;
3) Insert mixture into a PCR thermal cycler, design the PCR program according to the manufacturer's instructions.
4) Perform agarose gel electrophoresis, and the presence of the correct bands indicates the successful construction of recombination plasmids.
3.Protein expression and purification
3.1 Induction of protein expression for 6xHis-NF-L, 6xHis-Aβ-42, 6xHis-α-syn, 6xHis-NF-L-mcherry, 6xHis-Aβ-42-mcherry, and 6xHis-α-syn-mcherry
Ⅰ. Amplification culture for correct colony
Goal: To acquire large amounts of target proteins for SDS-PAGE
Materials:
1) Liquid LB solution
2) pET-28a(+)-NF-L/Aβ-42/α-syn and pET-28a(+)-NF-L/Aβ-42/α-syn-mcherry bacterial fluid
3) Kanamycin antibiotics
4) Inducer: IPTG
5) Iso-thermic shaker
Procedures:
1) Add 100μl of pET-28a(+)-NF-L/Aβ-42/α-syn and pET-28a(+)-NF-L/Aβ-42/α-syn-mcherry bacterial fluid and 100μl of Kanamycin antibiotics into 100mL of liquid LB solutions, respectively;
2) Culture bacterial liquid in iso-thermic shaker for 3 hours until absorbance OD600nm to 0.4-0.6;
3) Add IPTG into bacterial liquid with 1mM final concentration;
4) Sample for each bacterial liquid cultured at 25°C and 37°C for 0.5h, 1h, 3h, and 6h, respectively.
Ⅱ: Protein crude extraction and SDS-PAGE
Goal: To verify the expression of desired proteins.
Materials for protein crude extraction:
1) 500μl bacterial liquid for different induced conditions
2) Centrifuge
3) Double distilled water (ddH2O)
4) 6x Protein loading buffer
Procedures for protein crude extraction:
1) Centrifuge samples at 12000 rpm for 1 minute;
2) Discard supernatant and resuspend precipitation with 50μl 1x Protein loading buffer diluted by ddH2O;
3) Boiled samples at 95°C for 15 minutes in PCR thermal cycler.
Materials for SDS-PAGE:
1) Sangon 12.5% SDS-PAGE Color Preparation kit
2) Electrophoresis buffer (Tris-Glycine)
3) Protein ladder
4) 6x Protein loading buffer
5) Vertical electrophoresis system
6) Coomassie blue staining solution
Procedures for SDS-PAGE：
1) Add and mix 2.2mL 2x separating gel solution, 2.2mL 2X separating gel buffer, and 44μL catalyst in centrifugation tubes for separating gel;
2) Slowly pipetting mixture into casting stand and frame to avoid bubbles;
3) Add 1 mL ddH2O at the top of separating gel;
4) Wait the separating gel to solidify for 8 minutes;
5) Discard the water that was added previously;
6) Add and mix 825μL 2x stacking gel solution, 825μL 2X stacking gel buffer, and 11μL catalyst in centrifugation tubes for stacking gel;
7) Add stacking gel mixture until the cast is completely filled, then slowly insert comb without producing any air bubbles;
8) Wait the stacking gel to solidify for 12 minutes, then carefully remove the comb, and wash the wells with electrophoresis buffer;
9) Add 10μL protein ladder into the first well, then load samples in each successive well;
10) Transfer gel into vertical electrophoresis system;
11) Run at 120V for 90 minutes;
12) Stain the gel with Coomassie blue staining solution for 10 minutes, then destain the gel using destaining solution repeatedly until gel background become transparent.
Ⅲ: Western blotting for target proteins using anti-His tag antibody
Goal: To verify the correctness of recombination proteins by Western blotting, to calculate the relative gray intensity value of protein bands and finally to confirm the best induced conditions for target proteins.
Materials:
1) SDS-PAGE gel
2) Transfer buffer (Tris-Glycine added methanol)
3) PVDF membrane
4) TBST buffer (Tris-buffered saline with Tween-20)
5) Blocking buffer (5% non-fat dry milk in TBST)
6) Anti-His tag primary antibody from mouse
7) Goat anti-mouse HRP-conjugated secondary antibody
8) Chemiluminescent substrate
9) Imaging system
Procedures:
1) Gel electrophoresis: same as SDS-PAGE described at step 3.1 II;
2) After electrophoresis, carefully remove the gel and equilibrate it in transfer buffer for 10-15 minutes;
3) Assemble the transfer sandwich: place the PVDF membrane between the gel and filter papers in the transfer apparatus;
4) Transfer proteins from the gel to the PVDF membrane at 100 V for 2 hours on ice;
5) After transfer, rinse the membrane briefly with TBST;
6) Block non-specific binding sites by incubating the membrane in blocking buffer (5% non-fat dry milk in TBST) at room temperature for 1 hour;
7) Dilute the Anti-His tag primary antibody in blocking buffer for 1:5000;
8) Incubate the membrane with the diluted primary antibody at room temperature for 2 hours;
9) Wash the membrane 3 times with TBST (5 minutes each wash) to remove unbound antibodies;
10) Dilute the Goat anti-mouse HRP-conjugated secondary antibody in blocking buffer for 1:10000;
11) Incubate the membrane with the diluted secondary antibody at room temperature for 1 hours;
12) Wash the membrane 3 times with TBST (5 minutes each wash) to remove unbound secondary antibodies;
13) Prepare the chemiluminescent substrate A and B solutions according to the manufacturer's instructions;
14) Incubate the membrane with substrate solutions for 5 minutes until the membrane is fully covered;
15) Capture the signal using CCD imaging system to visualize the bands;
16) Analyze and calculate the relative gray intensity value of protein bands by ImageJ software.
3.2 Protein His-tag purification
Goal: To recover target proteins using His-tag and Ni-NTA beads affinity purification kit for antigen preparation.
Materials:
1) Bacteria culture containing desired protein
2) 50 mL centrifuge tubes
3) Beaker containing ice
4) Centrifuge
5) Sonicator
6) Beyotime Ni-NTA agarose beads purification kit
Procedures:
1) Add samples into 50 mL centrifuge tubes and then perform centrifugation at 4000 rpm for 20 minutes;
2) Put centrifuge tube into a beaker filled with ice and then insert the sonicator probe into centrifuge tube;
3) Sonicate for 15 minutes;
4) Repeat steps 2 and 3 for all samples;
5) Centrifugate samples at 10000 rpm for 30 minutes;
6) Remove cell fragments that have precipitated at the bottom of centrifuge tubes;
7) Transfer supernatant containing desired proteins to a new tube for remaining steps;
8) Add 500μl Ni-NTA agarose beads to a 2.0mL centrifuge tube, then centrifuge at 500g for 5 minutes;
9) Discard the supernatant, add 1.5mL PBS buffer to wash Ni-NTA agarose beads, and then centrifuge at 500g for 5 minutes;
10) Repeat step 9 for 3 times;
11) Add pre-treated Ni-NTA agarose beads to protein supernatant samples and then incubate at 4℃ for 60 minutes;
12) Add incubated protein to absorption column, stand for 2 minutes, and then drain the supernatant liquid;
13) Add 5mL Wash buffer to wash agarose beads;
14) Repeat step 13 for 5 times;
15) Add 500μl Elution buffer containing 250mM imidazole to elute protein;
16) Repeat step 15 for 5 times;
17) From step 13 to 16, collect all the fractions draining from absorption column;
18) Transfer 50μl sample and then add 6x Protein loading buffer;
19) Run and detect the purified protein by SDS-PAGE and Coomassie blue staining assay.
Ⅰ. Amplification culture for correct colony
Goal: To acquire large amounts of target proteins for SDS-PAGE
Materials:
1) Liquid LB solution
2) pET-28a(+)-NF-L/Aβ-42/α-syn and pET-28a(+)-NF-L/Aβ-42/α-syn-mcherry bacterial fluid
3) Kanamycin antibiotics
4) Inducer: IPTG
5) Iso-thermic shaker
Procedures:
1) Add 100μl of pET-28a(+)-NF-L/Aβ-42/α-syn and pET-28a(+)-NF-L/Aβ-42/α-syn-mcherry bacterial fluid and 100μl of Kanamycin antibiotics into 100mL of liquid LB solutions, respectively;
2) Culture bacterial liquid in iso-thermic shaker for 3 hours until absorbance OD600nm to 0.4-0.6;
3) Add IPTG into bacterial liquid with 1mM final concentration;
4) Sample for each bacterial liquid cultured at 25°C and 37°C for 0.5h, 1h, 3h, and 6h, respectively.
Ⅱ: Protein crude extraction and SDS-PAGE
Goal: To verify the expression of desired proteins.
Materials for protein crude extraction:
1) 500μl bacterial liquid for different induced conditions
2) Centrifuge
3) Double distilled water (ddH2O)
4) 6x Protein loading buffer
Procedures for protein crude extraction:
1) Centrifuge samples at 12000 rpm for 1 minute;
2) Discard supernatant and resuspend precipitation with 50μl 1x Protein loading buffer diluted by ddH2O;
3) Boiled samples at 95°C for 15 minutes in PCR thermal cycler.
Materials for SDS-PAGE:
1) Sangon 12.5% SDS-PAGE Color Preparation kit
2) Electrophoresis buffer (Tris-Glycine)
3) Protein ladder
4) 6x Protein loading buffer
5) Vertical electrophoresis system
6) Coomassie blue staining solution
Procedures for SDS-PAGE：
1) Add and mix 2.2mL 2x separating gel solution, 2.2mL 2X separating gel buffer, and 44μL catalyst in centrifugation tubes for separating gel;
2) Slowly pipetting mixture into casting stand and frame to avoid bubbles;
3) Add 1 mL ddH2O at the top of separating gel;
4) Wait the separating gel to solidify for 8 minutes;
5) Discard the water that was added previously;
6) Add and mix 825μL 2x stacking gel solution, 825μL 2X stacking gel buffer, and 11μL catalyst in centrifugation tubes for stacking gel;
7) Add stacking gel mixture until the cast is completely filled, then slowly insert comb without producing any air bubbles;
8) Wait the stacking gel to solidify for 12 minutes, then carefully remove the comb, and wash the wells with electrophoresis buffer;
9) Add 10μL protein ladder into the first well, then load samples in each successive well;
10) Transfer gel into vertical electrophoresis system;
11) Run at 120V for 90 minutes;
12) Stain the gel with Coomassie blue staining solution for 10 minutes, then destain the gel using destaining solution repeatedly until gel background become transparent.
Ⅲ: Western blotting for target proteins using anti-His tag antibody
Goal: To verify the correctness of recombination proteins by Western blotting, to calculate the relative gray intensity value of protein bands and finally to confirm the best induced conditions for target proteins.
Materials:
1) SDS-PAGE gel
2) Transfer buffer (Tris-Glycine added methanol)
3) PVDF membrane
4) TBST buffer (Tris-buffered saline with Tween-20)
5) Blocking buffer (5% non-fat dry milk in TBST)
6) Anti-His tag primary antibody from mouse
7) Goat anti-mouse HRP-conjugated secondary antibody
8) Chemiluminescent substrate
9) Imaging system
Procedures:
1) Gel electrophoresis: same as SDS-PAGE described at step 3.1 II;
2) After electrophoresis, carefully remove the gel and equilibrate it in transfer buffer for 10-15 minutes;
3) Assemble the transfer sandwich: place the PVDF membrane between the gel and filter papers in the transfer apparatus;
4) Transfer proteins from the gel to the PVDF membrane at 100 V for 2 hours on ice;
5) After transfer, rinse the membrane briefly with TBST;
6) Block non-specific binding sites by incubating the membrane in blocking buffer (5% non-fat dry milk in TBST) at room temperature for 1 hour;
7) Dilute the Anti-His tag primary antibody in blocking buffer for 1:5000;
8) Incubate the membrane with the diluted primary antibody at room temperature for 2 hours;
9) Wash the membrane 3 times with TBST (5 minutes each wash) to remove unbound antibodies;
10) Dilute the Goat anti-mouse HRP-conjugated secondary antibody in blocking buffer for 1:10000;
11) Incubate the membrane with the diluted secondary antibody at room temperature for 1 hours;
12) Wash the membrane 3 times with TBST (5 minutes each wash) to remove unbound secondary antibodies;
13) Prepare the chemiluminescent substrate A and B solutions according to the manufacturer's instructions;
14) Incubate the membrane with substrate solutions for 5 minutes until the membrane is fully covered;
15) Capture the signal using CCD imaging system to visualize the bands;
16) Analyze and calculate the relative gray intensity value of protein bands by ImageJ software.
3.2 Protein His-tag purification
Goal: To recover target proteins using His-tag and Ni-NTA beads affinity purification kit for antigen preparation.
Materials:
1) Bacteria culture containing desired protein
2) 50 mL centrifuge tubes
3) Beaker containing ice
4) Centrifuge
5) Sonicator
6) Beyotime Ni-NTA agarose beads purification kit
Procedures:
1) Add samples into 50 mL centrifuge tubes and then perform centrifugation at 4000 rpm for 20 minutes;
2) Put centrifuge tube into a beaker filled with ice and then insert the sonicator probe into centrifuge tube;
3) Sonicate for 15 minutes;
4) Repeat steps 2 and 3 for all samples;
5) Centrifugate samples at 10000 rpm for 30 minutes;
6) Remove cell fragments that have precipitated at the bottom of centrifuge tubes;
7) Transfer supernatant containing desired proteins to a new tube for remaining steps;
8) Add 500μl Ni-NTA agarose beads to a 2.0mL centrifuge tube, then centrifuge at 500g for 5 minutes;
9) Discard the supernatant, add 1.5mL PBS buffer to wash Ni-NTA agarose beads, and then centrifuge at 500g for 5 minutes;
10) Repeat step 9 for 3 times;
11) Add pre-treated Ni-NTA agarose beads to protein supernatant samples and then incubate at 4℃ for 60 minutes;
12) Add incubated protein to absorption column, stand for 2 minutes, and then drain the supernatant liquid;
13) Add 5mL Wash buffer to wash agarose beads;
14) Repeat step 13 for 5 times;
15) Add 500μl Elution buffer containing 250mM imidazole to elute protein;
16) Repeat step 15 for 5 times;
17) From step 13 to 16, collect all the fractions draining from absorption column;
18) Transfer 50μl sample and then add 6x Protein loading buffer;
19) Run and detect the purified protein by SDS-PAGE and Coomassie blue staining assay.
4. Functional testing
4.1 Determination of protein fused with mcherry tag by fluorescence microscope
Goal: To test the effectiveness of NF-L/Aβ-42/α-syn antigens using corresponding sandwich ELISA kits.
Materials:
1) Purified NF-L/Aβ-42/α-syn proteins using His-tag purification kits
2) Sandwich ELISA kits for NF-L
3) Sandwich ELISA kits for Aβ-42
4) Sandwich ELISA kits for α-syn
5) Coating PBS buffer
6) Blocking buffer
7) Sample diluent
8) Standard sample
9) Substrate solution
10) Wash buffer
11) Detection antibody conjugated to HRP
12) Stop solution (sulfuric acid for HRP)
13) Microplate Reader
Procedures:
1) Dilute Purified NF-L/Aβ-42/α-syn proteins in sample diluent as necessary;
2) Dilute standard samples in sample diluent according to the instruction;
3) Add 100 µL of each diluted sample and standard samples to 96-well microplate;
4) Cover and incubate for 1-2 hours at room temperature;
5) Wash the wells 3-5 times with washing buffer;
6) Prepare a dilution of the detection antibody in sample diluent;
7) Add 100 µL of the diluted detection antibody to each well;
8) Incubate for 1-2 hours at room temperature;
9) Wash the wells 3-5 times with washing buffer;
10) Add 100 µL of the substrate solution to each well. This substrate solution will react with HRP enzyme linked to the detection antibody;
11) Allow the color to develop for 30 minutes at room temperature in the dark;
12) Add 50 µL of stop solution to each well to terminate the enzymatic reaction;
13) Measure the absorbance of each well at 450 nm wavelength using microplate reader;
14) Determine the concentration of the target antigen in samples based on their absorbance values using the standard curve.
Goal: To test the effectiveness of NF-L/Aβ-42/α-syn antigens using corresponding sandwich ELISA kits.
Materials:
1) Purified NF-L/Aβ-42/α-syn proteins using His-tag purification kits
2) Sandwich ELISA kits for NF-L
3) Sandwich ELISA kits for Aβ-42
4) Sandwich ELISA kits for α-syn
5) Coating PBS buffer
6) Blocking buffer
7) Sample diluent
8) Standard sample
9) Substrate solution
10) Wash buffer
11) Detection antibody conjugated to HRP
12) Stop solution (sulfuric acid for HRP)
13) Microplate Reader
Procedures:
1) Dilute Purified NF-L/Aβ-42/α-syn proteins in sample diluent as necessary;
2) Dilute standard samples in sample diluent according to the instruction;
3) Add 100 µL of each diluted sample and standard samples to 96-well microplate;
4) Cover and incubate for 1-2 hours at room temperature;
5) Wash the wells 3-5 times with washing buffer;
6) Prepare a dilution of the detection antibody in sample diluent;
7) Add 100 µL of the diluted detection antibody to each well;
8) Incubate for 1-2 hours at room temperature;
9) Wash the wells 3-5 times with washing buffer;
10) Add 100 µL of the substrate solution to each well. This substrate solution will react with HRP enzyme linked to the detection antibody;
11) Allow the color to develop for 30 minutes at room temperature in the dark;
12) Add 50 µL of stop solution to each well to terminate the enzymatic reaction;
13) Measure the absorbance of each well at 450 nm wavelength using microplate reader;
14) Determine the concentration of the target antigen in samples based on their absorbance values using the standard curve.