1. DNA&RNA
1.1. DNA REPLICATION, DNA RECOMBINATION, RNA SPLICING OR EDITING
2. sample preparation
2.1. cell disruption
2.1.1. To release proteins from within cell
2.1.1.1. Mechanical method
2.1.1.2. Non-mechanical method
2.2. protein solubilization
2.2.1. To denature protein
2.2.2. To disrupt disulfide bond through reduction
2.2.3. To dissolve particles interfere with electrophoresis
2.2.3.1. Sample/Loading buffer
2.2.3.1.1. Tris-Cl
2.2.3.1.2. Glycerol
2.2.3.1.3. Sodium Dodecyl Sulfate (SDS)
2.2.3.1.4. B-mercaptoethanol
2.2.3.1.5. Bromophenol blue
2.3. contaminant removal
2.3.1. Remove interfering substances that can negatively impact SDS-PAGE
2.3.1.1. Buffer Exchange (Desalting)
2.3.1.2. Protein Precipitation ( Help concentrate sample if needed)
2.4. quantitation
2.4.1. BCA protein assay : Lowry, Bradford, Biuret
2.4.1.1. Concentration by spectrofotometer
2.4.2. Related to H atom, Dalton
2.4.2.1. Molecular Weight Markers
2.4.2.2. Migration pattern : gel type, gel concentration, running buffer
2.5. PAGE preparation
2.5.1. Dilute sample in appropriate sample buffer to a final sample buffer concentration of 1X
3. Size estimation
3.1. Distance migrate in mm (x-axis) against molecular mass of prestained protein band in kD (y-axis)
3.1.1. Linear Graph