User:Michael F. Nagle/Notebook/Chem 571/2012/09/26

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Objectives

  • Purify ADA via Fast Protein Liquid Chromatography

Procedure

  1. The machine was baselined with 25mL binding buffer in one tube and 25mL eluting buffer in another.
  2. The flow rate was 5 mL/min and pump flow was 0.15 MPa.
    1. The column was eluted with the binding buffer, then eluted with elution buffer, and again with the binding buffer to wash out excess imidazole.
  3. ADA was inserted and eluted with the binding buffer. The collumn was eluted with the eluting buffer, which contains imidazole, because it competes for bonds with the nickel, forcing off the ADA. The ADA were collected in 11 5mL tubes. Tubes for each of the two sets of protein were combined and refrigerated.

Data

These absorbance peaks show the amount of ADA in each tube. Image:ADA HisTrap2.PNG

Discussion

  • ADA has a polyhistidine tag, which is a motif of at least 5 histidine residues. The column is nickel because proteins including ADA with polyhistidine tags have high affinities for it. Imidazole has the same functional groups as the histidine residues and they both show absorbancy peaks at 280nm. The machine was baselined so that imidazole's peaks would interfere with ADA's.
  • The spectra show that most ADA was collected in the second tube for each sample.



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