According to previous method of serial affinity chromatography [33 (link)], 1 ml lysates of PANC-1 cells were gently stirred with 100 μl DMAPT-affinity probe or blank matrices (EAH-sepharose 4B) at 4 °C for approximately 40 min and then precipitated by centrifugation at 8,000 × g for 3 min. The supernatants were mixed with another 100 μl of DMAPT-affinity probe or blank matrices at 4 °C, and incubated for approximately 40 min, which was repeated for four times. The resulting resins were successively washed with lysate buffer and mixed with 40 μl of SDS loading buffer, boiled at 100 °C for 5 min, and then centrifuged for 3 min. The supernatants were subjected to SDS-PAGE. After silver staining of the gels, the protein bands were comparatively analyzed to identify the reduction in amount between each series for specific binding proteins. Individual protein band on the SDS-PAGE was excised and digested with trypsin. Tryptic peptides were analyzed on an Ultraflex II MALDI-TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) in a positive-ionization mode over the m/z range of 700-4,000 at a resolution of 15,000 to 20,000. Protein sequences were analyzed using the peptide mass fingerprint by Mascot Search engine (version 2.4) as previously reported [34 (link)].
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