Difference between revisions of "Part:BBa K3573001:Experience"
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We have tested protein expression in 4 different conditions (2 temperatures X 2 IPTG concentrations). After expression, 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, Ni-NTA column was used for protein purification using the His6-tag located at the N-terminus of CPP-scFv(RAS). | ||
| − | [[File:T--Korea_HS--scFvNiNTA.jpg| | + | [[File:T--Korea_HS--scFvNiNTA.jpg|800px]] |
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HRas(G12V) that is described part number BBa_K3573002 was used for binding assay with scFv(RAS) using size-exclusion chromatography. | HRas(G12V) that is described part number BBa_K3573002 was used for binding assay with scFv(RAS) using size-exclusion chromatography. | ||
| − | [[File:T--Korea_HS--scFvRASBinding.jpg| | + | Korea_HS team have performed binding assays using size-exclusion chromatography. We ran three separate runs of 3 superdex75 10/300GL column with buffer (20mM Tris-HCl pH 7.0+250mM NaCl) |
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| + | 1. scFv(RAS) only | ||
| + | 2. HRAS only | ||
| + | 3. scFv(RAS)+HRAS | ||
| + | |||
| + | The result showed that HRAS's elution peak shifted to higher molecular weight position in the presence of scFv(RAS) compared to HRAS only, indicating their binding. | ||
| + | |||
| + | [[File:T--Korea_HS--scFvRASBinding.jpg|800px]] | ||
===User Reviews=== | ===User Reviews=== | ||
Latest revision as of 00:02, 26 October 2020
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Applications of BBa_K3573001
Our team has amplified scFv(RAS) using PCR and cloned the gene into an expression vector.
We have tested protein expression in 4 different conditions (2 temperatures X 2 IPTG concentrations). After expression, Ni-NTA column was used for protein purification using the His6-tag located at the N-terminus of CPP-scFv(RAS).
We have confirmed the identity of the protein using Western blot analysis using anti-His6 antibody.
HRas(G12V) that is described part number BBa_K3573002 was used for binding assay with scFv(RAS) using size-exclusion chromatography.
Korea_HS team have performed binding assays using size-exclusion chromatography. We ran three separate runs of 3 superdex75 10/300GL column with buffer (20mM Tris-HCl pH 7.0+250mM NaCl)
1. scFv(RAS) only 2. HRAS only 3. scFv(RAS)+HRAS
The result showed that HRAS's elution peak shifted to higher molecular weight position in the presence of scFv(RAS) compared to HRAS only, indicating their binding.
User Reviews
UNIQ574c133a2965eb08-partinfo-00000000-QINU UNIQ574c133a2965eb08-partinfo-00000001-QINU