Difference between revisions of "Part:BBa K1456006:Experience"
(→‎Aprotinin Assay)
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==Aprotinin Assay==
 
==Aprotinin Assay==
 
<li>As Aprotinin is a serine protease inhibitor aprotinin activity can be measured using the correlation of serine protease inhibition the aprotinin inhibits.
 
<li>As Aprotinin is a serine protease inhibitor aprotinin activity can be measured using the correlation of serine protease inhibition the aprotinin inhibits.
−
<center>[[File:Atoms aprotinin 3.png|400px|]]</center><br/>
+
<center>[[File:Atoms aprotinin 3.png|600px|]]</center><br/>
 
<li>HEK 293T cell was transfected with 1 µg pTRE-Aprotinin and cells were obtained after 36 hours which continued with the isolation of proteins. Afterwards, the isolated protein was placed into a tube containing the serine protease and its activity was measured.
 
<li>HEK 293T cell was transfected with 1 µg pTRE-Aprotinin and cells were obtained after 36 hours which continued with the isolation of proteins. Afterwards, the isolated protein was placed into a tube containing the serine protease and its activity was measured.
 
<li>The graphic shows that serine protease inhibition was not observed in the isolated proteins of the cells transfected with the pTRE vectors and our negative controls only.
 
<li>The graphic shows that serine protease inhibition was not observed in the isolated proteins of the cells transfected with the pTRE vectors and our negative controls only.
 
<li>The inhibition values obtained by adding 6 mg/dl Bovine Aprotinin(sigmaA1153) samples obtained by cattles and the cotransfected Tet off – pTRE Aprotinin, show that the transfected HEK 293 T cells perform a lower amount of inhibition compared to our standard sample however that is a very minimal difference. As a result, our transfected aprotinin has accomplished more inhibition than our negative control and has proved that it can pe produced functionally. Afterwards the HEK 293 T cells cotransfected with Tet off – pTRE Aprotinin have been transfected in different amounts to characterise and measure its serine protease activity.
 
<li>The inhibition values obtained by adding 6 mg/dl Bovine Aprotinin(sigmaA1153) samples obtained by cattles and the cotransfected Tet off – pTRE Aprotinin, show that the transfected HEK 293 T cells perform a lower amount of inhibition compared to our standard sample however that is a very minimal difference. As a result, our transfected aprotinin has accomplished more inhibition than our negative control and has proved that it can pe produced functionally. Afterwards the HEK 293 T cells cotransfected with Tet off – pTRE Aprotinin have been transfected in different amounts to characterise and measure its serine protease activity.
 +
 
==Chacarterisation==
 
==Chacarterisation==
 
<center>[[File:Atoms aprotinin 4.png|400px|]]</center><br/>
 
<center>[[File:Atoms aprotinin 4.png|400px|]]</center><br/>

Revision as of 14:49, 21 October 2014

Aprotinin Assay

  • As Aprotinin is a serine protease inhibitor aprotinin activity can be measured using the correlation of serine protease inhibition the aprotinin inhibits.
    Atoms aprotinin 3.png

  • HEK 293T cell was transfected with 1 µg pTRE-Aprotinin and cells were obtained after 36 hours which continued with the isolation of proteins. Afterwards, the isolated protein was placed into a tube containing the serine protease and its activity was measured.
  • The graphic shows that serine protease inhibition was not observed in the isolated proteins of the cells transfected with the pTRE vectors and our negative controls only.
  • The inhibition values obtained by adding 6 mg/dl Bovine Aprotinin(sigmaA1153) samples obtained by cattles and the cotransfected Tet off – pTRE Aprotinin, show that the transfected HEK 293 T cells perform a lower amount of inhibition compared to our standard sample however that is a very minimal difference. As a result, our transfected aprotinin has accomplished more inhibition than our negative control and has proved that it can pe produced functionally. Afterwards the HEK 293 T cells cotransfected with Tet off – pTRE Aprotinin have been transfected in different amounts to characterise and measure its serine protease activity.

    Chacarterisation

    Atoms aprotinin 4.png

  • HEK 293T cells have been cotransfected with 1 µg, 2 µg, 3µg ve 4 µg Tet off – pTRE Aprotinin. The cells collected and protein isolation was performed to measure the serine protease inhibition. According to the positive control test, these results Show dose-dependent reduction and the production of aprotinin has increased according to the amount of cells successfully transfected.

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