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EXPERIMENT

2019/04/28 Protocol & Experiment

Creating recombinant gene:

Introduction

Custom-ordered synthesized gene pUCIDT vector includes scFv (P5), the cell-penetrating peptide that we are going to use.

The vector we are going to use is the pET-28a (+). Both pET-28a (+) and the pUCIDT vector have Nco I and Sal I restriction enzyme cut sites.

Protocol

1. Cut the scFv (P5) CPP from the pUCIDT vector using

Nco I and Sal I restriction enzyme

2. Cut the pET-28a (+) vector and insert the scFv (P5) CPP

by using sticky end ligase in a process called ligation

3. Make agarose gel for DNA electrophoresis

4. Check the combined vector with DNA gel electrophoresis

2019/05/26 Protocol & Experiment

Transformation and Expression of the recombinant plasmid (pET28b+-scFv(P5)) to e.coli competent cell(BL21(DE3)):

Introduction:

Competent cell: BL21(DE3)

B strains are deficient in Lon protease (cytoplasm) and OmpT protease (outer membrane). Accordingly, B strains are generally preferred for recombinant protein expression. The DE3 strains carries the gene for T7 RNA polymerase under 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.

Protocol

1. Competent cell 20㎕ + DNA(pET28b+-scFv(P5)) 1.5㎕

2. 20min in ice

3. 42℃ x 1min heat shock

4. 20min in ice

5. Add 200㎕ LB, shaking incubation 37℃ x 1Hr, 200rpm

6. Pre-heat the labelled plate

7. 3,000rpm x 3min table centrifuge

8. Discard 150㎕ of supernatant

9. Resuspension by tapping

10. Plating by streaking

11. Incubation 37℃, O/N

12. Check colony a day after

2019/06/09 Protocol & Experiment

Purification of histidine-tagged scFv(P5) using Ni2+–NTA resin affinity chromatography:

Introduction

Ni-NTA resin:

The Ni-NTA Resin is specifically designed for the purification of recombinant proteins fused to the 6xhistidine (6XHis) tag expressed in bacterial cells. The resin is high affinity and selectivity for recombinant fusion proteins that are tagged with six tandem histidine residues. Proteins bound to the resin can be eluted by competition with imidazole. NTA (nitrilotriacetic acid), binds with Ni2+ ions, acts as a chelating agent to keep the complex stable and water-soluble.

SDS-PAGE:

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 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.

Protocol

Ni2+–NTA resin affinity chromatography

Preparing soluble fraction of cell lysate. –SOL sample collect (10㎕)
Equilibration: Tris-HCl buffer 20mL.
Loading through: Soluble fraction of cell lysate –LT sample collect (10㎕)
Wash: 20mM imidazole 20mL. -WA sample collect (10㎕)
Elution 1: 100mM imidazole 5mL. -100mM sample collect (20㎕)
Elution 2: 400mM imidazole 5mL. -400mM sample collect (20㎕)

Preparing SDS-PAGE loading samples

Add 3’DW 10㎕ + 6x dye 4㎕ for SOL, LT samples

Add 6x dye 4㎕ for WA, 100mM, 400mM samples

Boiling for 5 minutes.

Check the scFv(P5) band through SDS-PAGE

2019/06/23 Protocol & Experiment

Identification:

Introduction

Western blot

The western blot is a widely used analytical

technique in molecular biology, immunogenetics

and other molecular biology disciplines to detect

specific proteins in a sample of tissue homogenate

or extract.

In brief, the sample undergoes protein denaturation,

followed by gel electrophoresis. A synthetic or

animal-derived antibody (known as the primary

antibody) is created that recognizes and binds to

a specific target protein. The electrophoresis

membrane is washed in a solution containing the

primary antibody, before excess antibody is washed

off. A secondary antibody is added which recognizes

and binds to the primary antibody. The secondary

antibody is visualized through various methods such

as staining, immunofluorescence, and radioactivity,

allowing indirect detection of the specific target

protein.

Procedure

SDS-PAGE → Transfer gel to PVDF membrane → Primary antibody incubation (6/23 experiment)

→ Secondary antibody incubation → Detection (7/7 experiment)

Protocol

Preparing SDS-PAGE loading samples

For 10㎕ of SOL, INS, LT, add 3’DW 10㎕ + 6x dye 4㎕.

For 20㎕ of WA, 100mM, 400mM, add 6x dye 4㎕.

For 5㎕ of positive control, add 3’DW 15㎕ + 6x dye 4㎕.

Boil for 5 minutes.

Transfer gel to PVDF membrane

1. Activate PVDF membrane with methanol for 1 min.

2. Soak filter papers and sponges in transfer buffer.

3. Prepare the stack as follows:

4. 100V, 90min with icepack.

5. Soak PVDF in methanol for 1 min, then dry the membrane.

Primary antibody incubation
20mL of 5% skim milk with 4㎕ of mouse-a-His6 antibody.

→ 4℃ incubation, O/N.

2019/07/07 Protocol & Experiment

Identification continued:

Protocol

Primary antibody wash (6/24)
10mL of TBS-T buffer x10min x3 times → -20℃ storage.

Secondary antibody incubation
20mL of 5% skim milk with 5㎕ of goat anti-mouse antibody → 1H incubation at room temp.

Secondary antibody wash
10mL of TBS-T buffer x10min x3 times.

→ Develop!

Binding Assay:

Introduction

Protein binding assay using SEC

Size exclusion chromatography

SEC separates molecules according to differences in size as they pass through a SEC medium packed in a column. Unlike IEX(Ion exchange) or AC(affinity chromatography), molecules do not bind to the chromatography medium so buffer composition does not directly affect resolution (the degree of separation between peaks). Consequently, a significant advantage of SEC is that conditions can be varied to suit the type of sample or the requirements for further purification, analysis, or storage without altering the separation.

To perform a separation, the medium is packed into a column to form a packed bed. SEC media consist of a porous matrix of spherical particles with chemical and physical stability and inertness (lack of reactivity and adsorptive properties). The packed bed is equilibrated with buffer, which fills the pores of the matrix and the space between the particles. The liquid inside the pores, or stationary phase, is in equilibrium with the liquid outside the particles, or mobile phase. Samples are eluted isocratically so there is no need to use different buffers during the separation. However, a wash step using the running buffer is usually included at the end of a separation to remove molecules that might have been retained on the column and to prepare the column for a new run.

Protocol

Sample Preparation

(Buffer: 20 mM Tris-HCl(pH7.5) + 250mM NaCl)

scFv sample: scFv 50㎕ + buffer 50㎕

lysozyme sample: lysozyme 50㎕ + buffer 50㎕

scFv + lysozyme sample: scFv + lysozyme 50㎕ each + buffer 10㎕

Size exclusion chromatography
(Filtered buffer: 20 mM Tris-HCl(pH7.5) + 250mM NaCl + 50mM DTT)

(Packing) Fill the column with resin resuspended in buffer. Avoid introducing air into the column.
(Equilibration) Inject 25mL buffer slowly.
(Sample loading) After equilibration, inject the sample.
(Elution) Add 1 column volume of buffer and collect 0.5ml fractions using capless tubes.

2019/07/14 Protocol & Experiment

2019/07/21 Protocol & Experiment

eGFP expression measurement experiment:

Protocol

1. Transform the eGFP gene contained plasmid to e.coli(Rosetta(DE3)) and plate on LB plates with antibiotics(carbenicillin). Grow overnight at 37℃.

2. Starter culture preparation: Prepare 40ml of LB with antibiotics and inoculate a single colony. Grow overnight at 37℃ with shaking.

3. Prepare regular flask and baffled flask with 500ml of LB. Seed 20ml of starter culture at each flask.

4. Grow at 37℃ with shaking. Keep measuring OD600.

5. When OD600 reaches 0.4~0.6, add 500㎕ of 1M isopropyl-β-thiogalactopyranoside(IPTG) to the cultures to induce expression.

6. Measure OD600 and fluorescence intensity every 15 minutes.

See RESULTS for clarity!

2019/08/11 Protocol & Experiment

eGFP expression measurement experiment:

We repeated the experiments done on July 21st, with only the change in measurement time. (from 20 minutes to 15 minutes.

Protocol

1. Transform the eGFP gene contained plasmid to e.coli(Rosetta(DE3)) and plate on LB plates with antibiotics(carbenicillin). Grow overnight at 37℃.

2. Starter culture preparation: Prepare 40ml of LB with antibiotics and inoculate a single colony. Grow overnight at 37℃ with shaking.

3. Prepare regular flask and baffled flask with 500ml of LB. Seed 20ml of starter culture at each flask.

4. Grow at 37℃ with shaking. Keep measuring OD600.

5. When OD600 reaches 0.4~0.6, add 500㎕ of 1M isopropyl-β-thiogalactopyranoside(IPTG) to the cultures to induce expression.

6. Measure OD600 and fluorescence intensity every 15 minutes.

See RESULTS