Fig. 3.

Diagram illustrating genesis of Protein A artefact in immunoaffinity purification procedure. Brain lysates were depleted of endogenous immunoglobulins using Protein A-conjugated Sepharose beads. During this step, some Protein A shed from the beads and contaminated the brain lysate. Upon exposure to the immunoaffinity matrix, some of the Protein A bound to the Fab region of the immobilized antibodies. This Protein A was then eluted from the matrix by the low pH elution buffer used to recover the target species. During subsequent Western blotting of the eluate from the antibody-conjugated magnetic beads, the denatured Protein A was still able to bind detection antibody