(A) E. coli competent cells (strain DH5α; TOYOBO, Japan) were freeze-thawed three times by freezing the cells on ice at −180°C then thawing them at 37°C. The lysates were then mixed with sample buffer lacking SDS and reducing agents and then subjected to PAGE analysis. One gel was immunoblotted with A11 antibody. A11-immunoreactive bands (A–G) were excised from the CBB-stained gel, treated with trypsin, and analyzed by tandem-mass spectrometry. A candidate A11-reactive protein from band B was GroEL. The difference in staining patterns between the A11 immunoblot and CBB-stained gel indicates A11 antibody-binding selectivity. Note also that the proteins in cell lysates reacted with the A11 antibody are not necessarily all natively folded. Purified GroEL (23 µM) was mixed with sample buffer lacking both reducing agents and SDS, and then run on (B) a gel in a regular SDS-containing buffer or (C) a gel in a buffer lacking SDS (native PAGE). Gels were blotted onto a membrane that was subsequently probed with A11 under the same conditions. Note that the mobility of the molecular weight standard is different in (B) and (C). (D) The following solutions (3 µL each) were dot blotted onto a nitrocellulose membrane: 20 µM Aβ40 monomer, 20 µM Aβ40 oligomer, H₂O, purified 23 µM GroEL, 37 µM Hsp27, 20 µM Hsp40, 14 µM Hsp70, 12 µM Hsp90, 10 µM Hsp104, and 10 µM Hsc70. GroEL, Hsp27, Hsp40, Hsp90, and Hsc70 were purchased from Stressgen (Canada). Hsp70 was from Sigma (USA). Hsp104 was from ATGen (South Korea). Each protein solution was blotted onto a single membrane that was probed with A11 under the same conditions. The individual dot blots were arranged into a single column.