Team:METU Turkey/Bravo

 

Purification with pTriEX vector

10/09/26
Objectives:
- 0.5 L culture of BL21 pTriEx-Rr-CooA French Pressed
- FPLC

Standard Experimental Parameters:

- Tris-HCI buffer is prepared again.
NOTE: As Temperature of Tris-HCl between 25-4 C decrease in 1 unit, pH of buffer increase in 0.003 unit.
- Buffer A:
20 mM Na2HPO4 [141.96 g/mol]
1 M NaCl [58.44 g/mol]
pH:7.2
- Buffer B :
Buffer A + Imidazole [6.81 g for 1L buffer A]
- Chelating column charged with zincsulfate-heptahydrate
[Applichem zincsulfate-heptahydrate A3439 ---- 287.54 g/mol]
1.44 g in 50 mL dH2O (100mM)
- Buffer A for chelating column 900 mL
- Buffer B for chelating column 900 mL
- Cleaning : 1 mL/min Buffer B 5 mL (5dk)
- Wash: 1 mL/min Buffer A 5 mL (5dk)
- 20 mL diluted sample
- 70 mL sample
- 0.5 mL elution tubes
- After FPLC results we combined the C13+C14+C15 tubes and C16+C17 tubes
Pooling fractions = [delay volume/fraction]+tube number
- SDS-Page

Purification with QS BL21 pTriEx

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This is 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 @ 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. According to FPLC graph, we have a sharp peak @ 280nm which contains proteins. We pick up the tubes between 9 and 14. SDS-Page shows the sharp peak 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.

Purification CS BL21 pTriEx

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Loaded combined FPLC tubes gave two close picks in CS column. First one corresponds to the CooA. The elutions of CooA are C13,C14 and C15. We combined them for further analyzes with EMSA and ITC and stored @+4. C16 AND C17 are combined to reload to CS column because they are contaminated another protein which is close to 40kDa.
10/09/30

Purification QS BL21 with pTriEx

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10/10/10
We desingned an experiment for characterization of CooA pTriEX vector.
We divided 1L culture into :
7 x 10 ml culture in 50 ml falcon x2 for IPTG concentration test
1 x 100 ml culture in 500 ml flask w/ Ferric Citrate
1 x 100 ml culture in 500 ml flask w/out Ferric Citrate
500 ml culture was used for purification of CooA
Our parameters was:
IPTG (1mM) w/ Ferric Citrate: 3/5/7/9 hours
IPTG (1mM) w/out Ferric Citrate: 3/5/7/9 hours
IPTG concentration: 0/0.05/0.5/0.1/0.25/0.5/1/2 mM @5 hours
For purification, we used general protein purification procedure.
Ferric concentration is 4mg/ml
We harvest 20 ml culture from 100ml flask for each IPTG (1mM) w/ Ferric Citrate and IPTG (1mM) w/out Ferric Citrate at time intervals 3-5-7-9. Then pellets was kept @-80 until sonication was performed. Pellets was sonicated then centrifugated at 20000g for 20 min. Supernatant was used for SDS-PAGE.
IPTG added to falcons: bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb
After 5 hours induction with different IPTG concentration, pellets was sonicated then centrifugated at 20000g for 20 min. Supernatant was used for SDS-PAGE.
We used french press for 500 ml Culture pellet BL21 pTriEx. We resuspended pellet in 50 ml Buffer A+ 25 ul protease inhibitor. Then Frech press and centrifugation in 32000 g for 60 min was done. The supernatant was used for SDS-PAGE.
10/10/10

Purification QS

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Purification QS cont. BB11
This gel photos indicate the elutes of 500ml culture which contains Bl21 with pTriEx. 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 combined @ 280,260 and 420 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

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This purification is done according to general protein purification procedure. BL21 was transformed with our pTriEx plasmid. We loaded 500 ml lik pellet to QS. We looked OD @ 260,280 and 420 according to the colors of the tubes. Yellowish color indicates our CooA protein. Then we looaded 27,28,29,3031,32,33 and 34 to SDS-Gel the elutes which have a high value of OD420 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. According to OD420 values, we combined the 7 tubes containing 1.5ml between 27-33,concentrated to 1ml and then diluted 10x with CS buffer A.
10/10/12 CS BB13 BB31
According to SDS-GEL, we combined 4,6,7,8 and 9 tubes containing 0,5ml and concentrated to 1ml for furhter analyzes such EMSA and ITF. We measured absorbance of combined @ 280,260 and 420 as respectively 2.76, 2.83 and 0.837. CooA concentration was determined as 7.75 uM according to extinction coefficient of heme(Ferric State) which is 108 mM-1 cm-1 according to Aono. We didnot take 5th tube because it contains much more impurities than other tubes.
10/10/21

Purification QS

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This purification is done according to general protein purification procedure. BL21 was transformed with our pTriEx plasmid. We loaded 500 ml lik pellet to QS. We looked OD @ 260,280 and 420 according to the colors of the tubes. Yellowish color indicates our CooA protein. Then we looaded 27,28,29,30 and 31 to SDS-Gel the elutes which have a high value of OD420 because oxidized CooA gives a sorret peak at 420 nm according to Aono.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.

Concentrated CooA

CooA protein contains heme group therefore its solution is seen yellow to orange BB16