Team:Korea HS/Results

KOREA_HS

Designing a Hyperstable Antibody with
Cell-penetrating Peptide for Intracellular Targeting

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