Difference between revisions of "Part:BBa K1202103:Experience"
Line 25: Line 25:
 
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
 
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.
 
monoclonal anti-HIS antibody.
 +
[[File:103-2.png]]
 
 
 
Using western blot analysis, it was observed that; our proteins expressed and purified effectively.
 
Using western blot analysis, it was observed that; our proteins expressed and purified effectively.

Revision as of 09:41, 9 October 2013

In this part of our project we tested are our signal peptides is working or not To secrete GFP protein via signal peptide to extracellular membrane. In overall project, our aim related with signal peptide is to secrete apoptotic proteins outer membrane. If this construct secrete GFP successfully, that means signal peptide works and TorA can be used in Apoptin or E4orf4 export. And also we use constitutive promoter in our designs because we need high amount of protein and we want to our bacteria always produce our proteins. We made santrifuge our signal peptides’ (TorA-GFP ve GFP-HlyA) liquid culture falcons and then get supernatant from falcons. Then we measured values of absorption of GFP in our supernatant in suitable wavelength. Results show that we found high amount of GFP in our supernatants. Result;

Sinyal.png

Column scale(y) shows us quantity amount of GFP in the solution.

we get good results from our GFP-HlyA that we found high amount of GFP in our samples more than our Negative(-) Control. Another good results that our Positive Control which is GFP that added in LB showed maximum amount of GFP in graphics. So we can said that our controls were working too. HLYA signal peptide results was like that.

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. 103-2.png

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


Applications of BBa_K1202103

User Reviews

UNIQb365ad8f41f9860f-partinfo-00000000-QINU UNIQb365ad8f41f9860f-partinfo-00000001-QINU