comparing between two protein with the same molecular mass and different shape .
SEC is usually used for separating molecules based on their size not shape, by gel filtration. The gel consists of spherical beads containing pores of a specific size distribution. Separation occurs when molecules of different sizes are included or excluded from the pores within the matrix. Small molecules diffuse into the pores and their flow through the column is retarded according to their size, while large molecules do not enter the pores and are eluted in the column's void volume. Consequently, molecules separate based on their size as they pass through the column and are eluted in order of decreasing molecular weight.
Well, SEC measures the hydrodynamic volume of the proteins, not really the mass, even though there is a relationship between the hydrodynamic volume and mass.
Personally, i have seen that proteins with same masses can be eluted in different volumes, and we think that this difference is due to their form.
Moreover, sometimes there may be some protein-matrix interactions that slighlty affect the retention time, thus changing the mass estimation.
So even though SEC is a very powerful method to separate proteins, for mass determination there may be some errors.
Hi there,
SEC column calibration is made with globular proteins. If YFP is not globular it will not obey the rule linking MW and retention time. And will tend to exhibit a bigger MW than its globular equivalent.
SEC is usually used for separating molecules based on their size not shape, by gel filtration. The gel consists of spherical beads containing pores of a specific size distribution. Separation occurs when molecules of different sizes are included or excluded from the pores within the matrix. Small molecules diffuse into the pores and their flow through the column is retarded according to their size, while large molecules do not enter the pores and are eluted in the column's void volume. Consequently, molecules separate based on their size as they pass through the column and are eluted in order of decreasing molecular weight.
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