Difference between revisions of "Team:Lethbridge HS/Results"

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Protein Purification</p>
 
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One of the major aspects of our system utilizes metal binding proteins, so it was imperative that we purify the protein in order to move forward and complete our Copper Binding Assay. Our team successfully purified the Cut A protein through Nickel Affinity Chromatography and Size Exclusion Chromatography.
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Figure 1 - 15% SDS-PAGE of Nickel Affinity batch purification of CutA.
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In the first lane we used the molecular weight marker RMR002 from GMbiolab. Lanes 2-5 show the elutions that contain our CutA protein. CutA protein runs at around 12kDA; however, in our SDS-PAGE gel it is seen at around 14kDa, this is likely because of the histidine tag. The remaining lanes are as follows: 6- Nickel Regeneration; 7- Cell Lysate Before Binding; 8- Cell Lysate After Binding; 9- Wash Sample; 10- Cell Pellet.
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The CutA protein was expressed in BL21 E.coli cells, and those cells were lysed then centrifuged to separate the supernatant and cell pellet. The lysate was then introduced to a Nickel Sepharose affinity column to isolate the CutA protein as it was bound to the Nickel Sepharose by its histidine tag. Then after washing to remove the unwanted proteins and cell debris, the CutA protein was eluted from the Nickel Sepharose.
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(Link to protocol)
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Revision as of 04:29, 17 October 2018



TITLE

Protein Purification

One of the major aspects of our system utilizes metal binding proteins, so it was imperative that we purify the protein in order to move forward and complete our Copper Binding Assay. Our team successfully purified the Cut A protein through Nickel Affinity Chromatography and Size Exclusion Chromatography. Figure 1 - 15% SDS-PAGE of Nickel Affinity batch purification of CutA. In the first lane we used the molecular weight marker RMR002 from GMbiolab. Lanes 2-5 show the elutions that contain our CutA protein. CutA protein runs at around 12kDA; however, in our SDS-PAGE gel it is seen at around 14kDa, this is likely because of the histidine tag. The remaining lanes are as follows: 6- Nickel Regeneration; 7- Cell Lysate Before Binding; 8- Cell Lysate After Binding; 9- Wash Sample; 10- Cell Pellet. The CutA protein was expressed in BL21 E.coli cells, and those cells were lysed then centrifuged to separate the supernatant and cell pellet. The lysate was then introduced to a Nickel Sepharose affinity column to isolate the CutA protein as it was bound to the Nickel Sepharose by its histidine tag. Then after washing to remove the unwanted proteins and cell debris, the CutA protein was eluted from the Nickel Sepharose. (Link to protocol)