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Cell Biology: A Laboratory Handbook

Affinity Purification with natural immobilized ligands

Publication ,  Chapter
Philip, N; Haystead, TA
November 16, 2005

This chapter discusses affinity chromatography-based techniques to examine protein interaction between other proteins or small ligands. The first procedure involves the use of toxin microcystin LR (MC-LR) conjugated to a biotin or Sepharose matrix to biochemically examine phosphatase-protein interactions. MC linked to biotin has the advantage of using mild conditions to elute bound proteins and also the holoenzyme components remain intact. Once the ATP-binding proteome is captured, various drugs are applied to the column. If the drug can compete with the natural ligand, the protein is eluted. Proteins are resolved by 1DE and identified by mass spectrometry. Proteome mining involves homogenizing 10 g of tissue in 40 ml of lyses buffer until mixture turns frothy. One needs to combine the supernatant with the ATP-Sepharose and rotate for 30 min at room temperature. MC-biotin has to be prepared fresh and is not reusable as it is lost during elution. It is important to know solubility conditions of the drugs used to elute proteins off the resin.

Duke Scholars

DOI

ISBN

9780121647308

Publication Date

November 16, 2005

Start / End Page

265 / 267
 

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Philip, N., & Haystead, T. A. (2005). Affinity Purification with natural immobilized ligands. In Cell Biology: A Laboratory Handbook (pp. 265–267). https://doi.org/10.1016/B978-012164730-8/50218-5
Philip, N., and T. A. Haystead. “Affinity Purification with natural immobilized ligands.” In Cell Biology: A Laboratory Handbook, 265–67, 2005. https://doi.org/10.1016/B978-012164730-8/50218-5.
Philip N, Haystead TA. Affinity Purification with natural immobilized ligands. In: Cell Biology: A Laboratory Handbook. 2005. p. 265–7.
Philip, N., and T. A. Haystead. “Affinity Purification with natural immobilized ligands.” Cell Biology: A Laboratory Handbook, 2005, pp. 265–67. Scopus, doi:10.1016/B978-012164730-8/50218-5.
Philip N, Haystead TA. Affinity Purification with natural immobilized ligands. Cell Biology: A Laboratory Handbook. 2005. p. 265–267.
Journal cover image

DOI

ISBN

9780121647308

Publication Date

November 16, 2005

Start / End Page

265 / 267