Designing a Hyperstable Antibody with
Cell-penetrating Peptide for Intracellular Targeting
5 July 2020
PCR amplification of insert (HRas G12V)
- PCR amplification of scFv(Ras) insert (805 bp)
- Set up PCR reactions and prepare to run PCR program
- PCR Mix
: Apply (50 μL total)
- 42 μL of sterile ddH2O
- 5 μL of reaction buffer (10x)
- Try Pfu first and if it does not work, try Vent
- 0.5 μL of dNTP mix at 2.5 mM
- 0.5 μL of forward primer at 100 μM
- 0.5 μL of reverse primer at 100 μM
- 1 μL of genomic or plasmid DNA
- 0.5 μL of DNA polymerase
- PCR Program
- Initial Denaturation at 94ºC – 5 min
- Denaturation at 94ºC – 1 min
- Annealing at 55ºC – 30 sec
- Extension at 72ºC – 1 min./kb target sequence
- Go to steps ii through iv again (25 times)
- Final Extension at 72ºC – 5 min
- Storage at 4ºC – ∞
- Check the PCR product with DNA gel electrophoresis.
- Dissolve 0.36 g agarose in 30 mL TAE buffer (1x)
- Microwave the mixture to a boil, and be careful not to boil over
- Cool down to a point below 60℃ and add 0.6 ㎕ of EtBr
- EtBr is very dangerous so make sure to wear gloves when handling the chemical
- Stir well and pour it to a gel cast
- Insert the comb in the gel cast and let the gel solidify for 30 minutes
- Remove the comb and apply samples from PCR
- Run gel electrophoresis for 15 min at 120V
- Check if HRas G12V insert (610 base pairs) is present
12 July 2020
Transformation and recombination of the plasmid (LIC vector - scFv(Ras)) to E. coli competent cell (BL21(DE)3)
B strains are deficient in Lon protease (cytoplasm) and OmpT protease (outer membrane). Therefore, B strains are generally preferred for recombinant protein expression. The DE3 strains carry the gene for T7 RNA polymerase under the control of the lacUV5 promoter. IPTG is required to maximally induce expression of the T7 RNA polymerase in order to express recombinant genes cloned downstream of a T7 promoter. BL21(DE3) is suitable for expression from a T7 or T7-lac promoter or promoters recognized by the E.coli RNA polymerase.
- Create a mixture using 20 ㎕ of competent cell and 1.5 ㎕ of DNA (LIC vector - scFv(Ras))
- Put the mixture in ice for 20 minutes
- Apply heat shock for 1 minute at 42℃
- Put the mixture back in ice for 20 minutes
- Add 200 ㎕ of LB, shaking incubation at 37℃ for one hour at 200 rpm
- Make sure to label and preheat LB plate
- Prepare a tabletop centrifuge and centrifuge for 3 minutes at 3,000 rpm
- Discard 150 ㎕ of the supernatant by pouring or using a pipette
- Resuspend the cell pellets by gentle tapping
- Start plating by streaking and put the plates in an incubator at 37℃ overnight
19 July 2020
Purification of histidine-tagged scFv(Ras) using Ni2+-NTA resin affinity chromatography
- Prepare soluble fraction of cell lysate –SOL sample collect (10 ㎕)
- Equilibration: 20 mM Tris-HCl pH 7.5 + 250 mM NaCl buffer 5CV (column volume)
- Loading through: soluble fraction of cell lysate –LT sample collect (10 ㎕)
- Wash: buffer + 20 mM imidazole 5CV –WA sample collect (20 ㎕)
- Elution 1: buffer + 100 mM imidazole 5mL - 100mM sample collect (20 ㎕)
- Elution 2: buffer + 400 mM imidazole 5mL - 400mM sample collect (20 ㎕)
23 July 2020
SDS-PAGE of purified histidine-tagged (scFv(Ras))
SDS-PAGE is a method of separating proteins based on their molecular mass. SDS(sodium dodecyl sulfate) is a detergent that binds proteins and covers them with a negative charge. After exposure to SDS different proteins will have a very similar charge to mass ratios because SDS coats the protein in a negative charge overwhelming whatever intrinsic charge the protein originally had. The denatured protein mixture is added to a polyacrylamide gel where an electric field is applied and the proteins move towards the positive electrode based on their size. The upper gel(stacking gel) first allows proteins to align the top of the resolving gel, then resolving gel separate proteins on their molecular weight.
- Preparing SDS-PAGE loading samples
- Add 3’DW 10 ㎕ + 6x dye 4 ㎕ for SOL, LT samples
- Add 6x dye 4 ㎕ for WA, 100 mM, 400 mM samples.
- Boil the sample for 5 minutes.
- Check the scFv(Ras) band through SDS-PAGE
- Preparing SDS-PAGE gel
- Assemble the gel casting apparatus, making sure that the sandwich of glass plates makes a good seal.
- Prepare 5 mL of separating gel solution according to the 12% acrylamide concentration recipe. Mix thoroughly.
- Fill the apparatus with separating gel solution, leaving about 2 cm of space for the stacking gel.
- Fill the apparatus with around 1 mL of 2-propanol to isolate the polymerization reaction from oxygen and flatten the separating gel.
- Give around 20 minutes for the separating gel to solidify.
- After the separating gel solidifies, pour off the 2-propanol and rinse with distilled water.
- Remove any water droplets from the inside of the casting apparatus with Whatman filter paper.
- Prepare 2 mL of 5% stacking gel solution. Mix thoroughly.
- Fill the stacking gel solution to the top. Place the 10-well comb into the apparatus without introducing air bubbles.
- Allow the stacking gel to polymerize completely for around 20 minutes before removing the comb.
- Prepare the samples
i) Mix 15 ㎕ protein samples with 3 ㎕ of SDS Sample Buffer (6x)
ii) Prepare the following samples (crude, loading through, wash, elution)
iii) Heat the samples for 5 minutes in boiling water
- Clip the sandwich to the electrophoresis apparatus. Carefully remove the comb from the gel and fill the inside of the apparatus with an SDS running buffer (1x).
- Carefully load 5 ㎕ of the protein MW marker and 10 ㎕ of samples (soluble, insoluble, loading thru, wash, 100 mM, 400 mM elution fractions) into the bottom of the wells using P20 pipette.
- Fill the outside of the electrophoresis apparatus with SDS running buffer (1x) and connect the apparatus to the power supply.
- Run the gel at 150V for around 60 minutes (one hour) until the dye reaches the bottom of the separating gel.
- Turn off the power supply, and remove the gel sandwich.
- Carefully open the sandwich by using one of the spacers to pry the plates apart.
- Put the gel in about 20 mL of Coomassie staining solution. Shake gently for at least 30 minutes.
- Pour the staining solution back to the bottle and destain the gel using around 100 mL of the destaining solution for at least 30 minutes.
- Pour off the destaining solution and put the gel in water.
- Take a picture of the gel
30 July 2020
- Prepare 1 L of 1X transfer buffer mix (680 ml dH2O + 170 ml 5X transfer buffer + 150 ml MeOH)
- Assemble the transfer sandwich (sponge – paper – gel – membrane – paper – sponge). Make sure the PVDF membrane should be placed on MeOH for ~1 min to activate and no air bubbles are trapped in the sandwich. The
- membrane should be on the cathode and the gel on the anode.
- Place the cassette in the transfer tank and place an ice block in the tank.
- Transfer the gel for 60 min at 100 V.
- Transferred membranes should be placed on MeOH and dry to remove residual alcohol.
- Incubate membrane in TBS-T buffer at 4 oC for the next experiment.
- Remove TBS-T buffer and incubate membrane in the primary antibody solution (4 ul anti-His6 antibody in 20 ml 5% milk) for 24 hours at 4 oC.
- Rinse the membrane 3 times for 10 min with TBS-T.
- Incubate membrane in the secondary antibody solution (5ul Goat anti-mouse IgG in 20 ml 5% milk) for 30 minutes at room temperature.
- Rinse the membrane 3 times for 10 min with TBS-T.
- Mix the Solution A and B at a 1:1 ratio to make PicoEPD western blot detection substrate working solution (1ml of working solution is sufficient).
- Wet the blotting membrane and cover the blot with clear plastic wrap.
- Expose the membrane to films in a dark room.
30 July 2020
Binding assay of scFv(Ras) and HRas
- Sample preparation
- Buffer: 20 mM Tris-HCl pH 7.5 + 250 mM NaCl
- scFv(Ras) sample: scFv(Ras) 250 ㎕ + buffer 250 ㎕
- HRas G12V sample: HRas G12V 250 ㎕ + buffer 250 ㎕
- scFv(Ras) + HRas G12V sample: scFv(Ras) 250 ㎕ + HRas G12V 250 ㎕
- size exclusion chromatography (filtered buffer: 20 mM Tris-HCl pH 7.5 + 250 mM NaCl)
- Equilibration: buffer 1CV
- Sample loading: after equilibration, inject the sample
- Elution: add 1 CV of buffer and collect 0.5 mL fractions using capless tube