Ther will be very small differences in base composition leading to slight variations in size because your samples will have a different base composition from that of the ladder but using the same ladder for different runs will give the same size for your product. You may get an apparently different size using a high molecular weight ladder from the size when using a low molecular weight ladder . You may have to accept that samples run at the extreme ends of the ladder sizes are likely to be inaccurate compared to samples running near the mid range of the ladder but samples will spread over a range of size in any gel run so reading off size against any ladder is only an estimate ( although a reproducible one) of size

Paul Rutland Thanks, dear sir.

Due to the large pore size of the upper gel, protein molecules of different sizes are basically blocked the same way when passing through the macroporous gel, so they move at the same rate; when entering the small pore gel, proteins with large molecular weights move slower.