Difference between revisions of "Part:BBa K3573001:Experience"

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===Applications of BBa_K3573001===
 
===Applications of BBa_K3573001===
  
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Our team has cloned scFv(RAS) into an expression vector and tested the protein expression in 4 different conditions (2 temperatures X 2 IPTG concentrations)
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Our team has amplified scFv(RAS) using PCR and cloned the gene into an expression vector.
  
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After expression, we used Ni-NTA column 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.
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[[File:T--Korea_HS--scFvPCR.jpg|500px]]
  
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[[File:T--Korea_HS--BBa_K3573001_1.png|500px]]
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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).
 +
 
 +
[[File:T--Korea_HS--scFvNiNTA.jpg|800px]]
 +
 
 +
 
 +
 
 +
We have confirmed the identity of the protein using Western blot analysis using anti-His6 antibody.
 +
 
 +
[[File:T--Korea_HS--scFvWestern.jpg|500px]]
 +
 
 +
 
 +
 
 +
HRas(G12V) that is described part number BBa_K3573002 was used for binding assay with scFv(RAS) using size-exclusion chromatography.
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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
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2. HRAS only
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3. scFv(RAS)+HRAS
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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.
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 +
[[File:T--Korea_HS--scFvRASBinding.jpg|800px]]
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 00:02, 26 October 2020


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Please enter how you used this part and how it worked out.

Applications of BBa_K3573001

Our team has amplified scFv(RAS) using PCR and cloned the gene into an expression vector.

T--Korea HS--scFvPCR.jpg


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).

T--Korea HS--scFvNiNTA.jpg


We have confirmed the identity of the protein using Western blot analysis using anti-His6 antibody.

T--Korea HS--scFvWestern.jpg


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.

T--Korea HS--scFvRASBinding.jpg

User Reviews

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