Skip to main content
Fig. 2 | Biological Procedures Online

Fig. 2

From: A Cautionary Tale: Endogenous Biotinylated Proteins and Exogenously-Introduced Protein A Cause Antibody-Independent Artefacts in Western Blot Studies of Brain-Derived Proteins

Fig. 2

Protein A contamination leads to artefactual bands during immunoaffinity purification. a Similar bands are detected by Western blot after immunoaffinity purification (anti-Aβ40/42 immunoaffinity matrix) of brain lysates from hAPP-J20 (J20) or non-transgenic (nTg) mice. Left, blot probed with 6E10-biotin, followed by IR-conjugated Neutravidin; right, blot probed with A11 followed by IR-conjugated goat-anti-rabbit IgG. Because monoclonal antibody 6E10 does not recognize mouse Aβ/APP, bands cannot represent genuine 6E10 immunoreactivity. b Molecular weights of bands detected by Western blot depend upon the source of Protein A used for immunodepletion. Brain lysates from J20 or nTg mice were depleted of endogenous immunoglobulins using either recombinant (recomb) or native Protein A (PrA), conjugated to Sepharose beads, and then subjected to anti-Aβ40/42 immunoaffinity purification. Eluates were analyzed in Western blots probed with 6E10 (left, green) or A11 (right, red). “B” marks material recovered after boiling of Protein A-conjugated Sepharose beads. c Bands present only in samples exposed to Protein A. Brain lysates from J20 or nTg mice underwent anti-Aβ40/42 immunoaffinity purification, either without prior depletion of endogenous immunoglobulins (N) or following depletion using Protein A (recombinant) and/or Protein G. Western blots of the eluates from the anti-Aβ40/42 matrix, probed with 6E10 (left, green) or A11 (right, red). “Beads” marks material recovered after boiling of Protein A- or Protein G- conjugated Sepharose beads. d Multiple antibodies react with denatured Protein A. Protein A-conjugated Sepharose beads were boiled in SDS-PAGE loading buffer, and recovered material was subjected to SDS-PAGE and Western blot. Blots were probed using a panel of antibodies (Table 1). e Brain lysates from J20 or nTg mice were subjected to single-step (anti-Aβ40/42) or sequential (6E10 followed by anti-Aβ40/42) immunoaffinity purification. IAC, immunoaffinity capture. nPrA ID, lysate immunodepleted using (native) Protein A-Sepharose prior to immunoaffinity purification; Y, yes; N, no. Blots probed with 6E10-biotin, followed by IR-conjugated Neutravidin. “B,” material recovered after boiling Protein A-Sepharose

Back to article page