Difference between revisions of "Part:BBa K1202106:Experience"

 
 
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    We controlled our genes in gel electrophoresis. Electrophoresis showed us the transformation of new genes was successful.There ise gel electrophoresis results below;
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[[File:103.png]]
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===TAT Apoptin purification experiment ; ===
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To purify the recombinant proteins, cells were spun down from 50 mL of culturesupernatant and resuspended in phosphate buffer saline.PBS contained PMSF in order to prevent proteis from proteases. The mixture was then sonicated on ice eight times for 30 second with a 52% pulsedactivity cycle (MISONIX SonicatorW 300). Next, the lysate was centrifuged for 30 min at 10,000 rpm to removethe cell debris. The resulting cell supernatant was loadedonto HIS select affinity column (Sİgma Aldrich)) for protein purification using the standard procedure.. The totalprotein concentration of each collected fraction fromthe column was determined using Bradford protein assay with bovine serum albumin actingas the reference protein. The purity of the protein fromeach fraction was analyzed by 12.5% SDS-PAGE andthen the resulting gels were Western blotted using
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monoclonal anti-HIS antibody.
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Using western blot analysis, it was observed that; our proteins expressed and purified effectively.
  
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__NOTOC__
 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
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how you used this part and how it worked out.
 
  
 
===Applications of BBa_K1202106===
 
===Applications of BBa_K1202106===

Latest revision as of 11:07, 9 October 2013

   We controlled our genes in gel electrophoresis. Electrophoresis showed us the transformation of new genes was successful.There ise gel electrophoresis results below;

103.png

TAT Apoptin purification experiment ;

To purify the recombinant proteins, cells were spun down from 50 mL of culturesupernatant and resuspended in phosphate buffer saline.PBS contained PMSF in order to prevent proteis from proteases. The mixture was then sonicated on ice eight times for 30 second with a 52% pulsedactivity cycle (MISONIX SonicatorW 300). Next, the lysate was centrifuged for 30 min at 10,000 rpm to removethe cell debris. The resulting cell supernatant was loadedonto HIS select affinity column (Sİgma Aldrich)) for protein purification using the standard procedure.. The totalprotein concentration of each collected fraction fromthe column was determined using Bradford protein assay with bovine serum albumin actingas the reference protein. The purity of the protein fromeach fraction was analyzed by 12.5% SDS-PAGE andthen the resulting gels were Western blotted using monoclonal anti-HIS antibody.


Using western blot analysis, it was observed that; our proteins expressed and purified effectively.


Applications of BBa_K1202106

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