For calculation of the Rf-values of SDS-proteins the separation distances of these molecules in relation to the Bromophenolblue (BPB) must be determined.
It is important to define the end of the stacking- and the beginning of the separation gel. This line can also slightly vary between different LOTs.
A SDS-protein micelle of more than 300 kD cannot enter a 12.5% separation gel. Due to this fact the parting line can be found by using the biggest SDS-standard protein of the “Prosieve QuadColor”: 315 kD - red color.
Migration distance of the BPB is then 1, the beginning of the separation gel is 0.
See page “SDS-Standards” and the pictures below.