What is the best buffer system and column for total cell protein extract? 2-D showing proteins ranging from 400KD to 0.2 KD size of proteins.
The best method for the determination of Ag. molecular wt. and size determination using HPLC is to employ a Size-Exclusion/GP Chromatography using MDS Detector which includes simultaneous detection through RI, LS and Viscometer. The pH, ionic strength and composition of the buffer usually, will not significantly affect resolution as long as they do not alter the size and stability of the protein to be separated. However, a low concentration of salt, between, 25 and 150 mM NaCl, should be used to reduce weak electrostatic interactions between proteins and the Gel Filtration media. The sample buffer does not have to be the same buffer as the column. The sample is exchanged into the running buffer during separation. Prior to using all buffers should
be filter through either a 0.45μm or a 0.22μm filter to eliminate debris, if any.
- Badges
- Science method