Agilent Protein Sizing and Quantitation with the Agilent Protein 80 and Protein 230 Kits on the Agilent 2100 Bioanalyzer 说明书用户手册.PDF

Protein Sizing and Quantitation with the Agilent Protein 80 and Protein 230 Kits on the Agilent 2100 Bioanalyzer Technical Overview Introduction The Agilent 2100 Bioanalyzer employs microfluidic capillary gel electrophoresis for protein analysis where fluorescence intensities of proteins are measured as a func- tion of their migration times. Data analysis is performed by the Agilent 2100 Expert Software, automatically determining molecular weight and concentration of the pro- teins in the sample. This Technical Overview describes the underlying mathematical operations for obtaining protein size and concentration information. Basic Principle of Protein Sizing and Quantitation The two key parameters for protein sizing and quantitation with the Agilent 2100 Bioanalyzer are migration time and fluorescence intensity. The migration time relates to the size of the proteins: larger proteins move slower due to the sieving effect of the gel inside the chip channels. Fluorescence intensity, in turn, is related to the concentration of these proteins, which are indirectly stained by a fluorescent dye that intercalates with protein-SDS-micelles. This dye is part of the sieving gel matrix, which is put onto the chip and reacts with proteins during separation. Since the migration time, as well as the fluorescence intensity, varies during the measurement of multiple samples, internal and external standards are used for cal- culating size and quantitation. The external standard is the specific protein ladder that is always analyzed on-chip before the actual sample measurements are done. Figure 1 Alignment of a P230 chip run. On the left the unaligned raw data is shown in the gel-like image representation. On the right, the same analysis after sample alignment based on lower marker (green) and upper marker (purple). Size and concentration are known for Figure 1 shows this effect on the left from the ladder, the external stand