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--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


This experience page is provided so that any user may enter their experience using this part.
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

UNIQb6aa74637f55788c-partinfo-00000000-QINU UNIQb6aa74637f55788c-partinfo-00000001-QINU