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

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==Procedure==
==Procedure==
#The machine was baselined with 25mL binding buffer in one tube and 25mL eluting buffer in another.
#The machine was baselined with 25mL binding buffer in one tube and 25mL eluting buffer in another.
##The collumn was eluted with the binding buffer
##The collumn was eluted with the binding buffer, then eluted with elution buffer, and again with the binding buffer to wash out excess imidazole.
##Collumn was eluted with elution buffer
#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 and protein were collected in 11 5mL tubes.
##Collum was eluted again with the binding buffer to wash out excess imidazole
 
##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 and protein were collected in 11 5mL tubes.
==Data==
==Data==
These absorbance peaks show the amount of ADA in each tube.
These absorbance peaks show the amount of ADA in each tube.

Revision as of 21:01, 4 October 2012

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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.
    1. The collumn was eluted with the binding buffer, then eluted with elution buffer, and again with the binding buffer to wash out excess imidazole.
  2. 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 and protein were collected in 11 5mL tubes.

Data

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

Discussion

  • The protein has a polyhistidine tag, which is a motif of at least 5 histidine residues. The collum 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 absorb

radiation 280nm. This increases the absorbance peaks for ADA, so that they can be measured.

  • The spectra show that most ADA was collected in the second tube for each sample.



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