Figure 4. Molecular weight determination of the BASS domain by mass spectrometry and native PAGE.
(a) Mass spectrum of the BASS domain measured on SYNAPT G2 HDMS under native-like conditions (b) Native PAGE was performed at eight different acrylamide concentrations for purified BASS (5 μg protein loaded into each lane) along with all the standards. Each lane represents a lane from a gel of different %T as indicated. The lanes were cut out from the original photos and aligned for comparison. (c) Standard curve drawn for an individual experiment. The relationship between the retardation coefficient (Kr) values determined for each standard protein and corresponding molecular weights were plotted in a logarithmic scale as a standard curve (R2=0.9856). The MW of BASS is marked with a red square on the standard curve. According to the result of three independent native PAGE experiments the MW of BASS is 153±16 kD. The inset shows a Ferguson plot for the BASS domain (semi logarithmic graph 100 × log(100 × Rf) versus %T). Rf and Log Rf values were multiplied by a factor of 100 to avoid negative values in the graphs. Graph shows the relationship between relative mobility of BASS and acrylamide concentration (R2=0.9905). The slope of the line represents Kr for BASS domain. The molecular weight standards as numbered in the Figure are given in the methods section.