Team:Korea HS/Results
KOREA_HS
Designing a Hyperstable Antibody with
Cell-penetrating Peptide for Intracellular Targeting
- Contribution
Results
1st Lab Results
PCR amplification of insert (HRas G12V)
A. PCR of HRas
Our team has amplified HRas using PCR and cloned the gene into an expression vector.
B. PCR of scFv(Ras)
Our team has amplified scFv(Ras) using PCR and cloned the gene into an expression vector.
2nd Lab Results
Transformation and recombination of the plasmid (LIC vector - scFv(Ras)) to E. coli competent cell (BL21(DE)3)
Above is a plate obtained from transformation.
3rd & 4th Lab Results
SDS-PAGE of purified histidine-tagged (scFv(Ras))
HRas (G12V) test expression
We have tested protein expression in 4 different conditions (2 temperatures X 2 IPTG concentrations). After expression, the Ni-NTA column was used for protein purification using the His6-tag located at the N-terminus of CPP-scFv(Ras).
We have tested protein expression in 4 different conditions (2 temperatures X 2 IPTG concentrations). After expression, the Ni-NTA column was used for protein purification using the His6-tag located at the N-terminus of CPP-scFv(Ras).
5th Lab Results
Western blotting
A. CPP-scFv(Ras) western blot using anti-His6 antibody
We have confirmed the identity of the protein using Western blot analysis using anti-His6 antibody.
B. HRas(G12V) western blot using anti-His6 antibody
To confirm the identity of the protein, we performed Western blot using anti-his antibody. The result showed our protein and positive control(pc) is detected.
Assay
Binding assay using size-exclusion column
Using a size-exclusion column, we tested if scFv(Ras) and HRas forms a complex. From comparisons of three runs (scFv only, HRas only and scFv÷HRas), shift of HRas peak to higher molecular weight is observed. The complex formation was also confirmed by SDS~PAGE analysis.
Summary
We were successful in:
- Amplifying HRas (G12V) and scFv(Ras) using PCR and cloning the gene into an expression vector.
- Testing protein expression in 4 different conditions
- Confirming the identity of the protein using Western blot analysis using anti-His6 antibody
- Confirming the complex formation using size-exclusion chromatography and SDS-PAGE analysis
Unfortunately, we were not successful in:
- His6 tag cutting using TEV protease. SDS-Page results were unclear and therefore we had to send it for N-term sequencing