Team:METU Turkey/Results Discussion/QS results

 

Purification with QS BL21 pTriEx
This was the first experiment of BL21 pTriEx with FPLC. We obeyed the general protein purification procedure except sonication. We lysed pellets with French Press. Then we centrifued the crude lysate at 32000 g for 60 min. We took a sample for SDS-PAGE. Then we continue with filtration. Filtered crude lysate was looaded to Q-sepharose column. According to FPLC graph, we have a sharp peak at 280nm which represents proteins. We picked up the tubes between 9 and 14. SDS-Page shows the sharp peak that contains CooA. F11 is the top of the sharp peak. We also check small peak which corresponds to 46th tube. We combined the F10, F11 and F12 elutes for further purification with chelating sepharose column.

10/09/30

Purification QS BL21 with pTriEx
We obeyed the general protein purification procedure. We took a sample for SDS-PAGE. Then we continue with filtration. Filtered crude lysate was looaded to Q-sepharose column. According to FPLC graph, we have a sharp peak at 280nm which represents proteins. We picked up the peak 3, peak 6 and peak 7. SDS-Page shows the sharp peak that contains CooA. We combined the elutes responsible from peak 3 for further purification with chelating sepharose column.

10/10/10
Purification QS with CS BL21 pTriEx

This gel photos indicate the elutes of 500ml culture which contains Bl21 with pTriEx vector. The crude lysate was loaded to Q-Sepharose column. In crude lysate, we observe the dimer and monomer forms of CooA. In Elute #27, we obtained high amount of CooA. We measured absorbance of elute 27 at 280,260 and 420 nm as respectively ?, ? and ?. CooA concentration was determined as ? uM according to extinction coefficient of heme (Ferric State) which is 108 mM-1 cm-1 according to Aono. We used it for further analyzes for EMSA and ITF.

10/10/12
Purification QS with CS BL21 pTriEx

This purification was done according to general protein purification procedure. BL21 was transformed with the pTriEx plasmid. We loaded 500 ml lik pellet to QS. We looked OD values at 260,280 and 420 according to the colors of the tubes. Yellowish to orange color indicates CooA protein. Then we looaded 27,28,29,30,31,32,33 and 34 to SDS-Gel, we selected the elutes according to their high value of 420nm because CooA gives a Sorret peak at 420 nm according to Aono. Oxidized CooA concentration was determined according to extinction coefficient of heme (Ferric State) which is 108 mM-1 cm-1 according to Aono. The SDS gel indicates that our protein is colose to 50 kDa in dimer. MW of monomer is estimated 24-25kDa according to Aono. As a results of the values of 420 nm of samples, we combined the 7 tubes containing 1.5ml between 27-33,concentrated to 1ml and then diluted 10x with CS buffer A.