Calibration plot for Hi-Load Superdex-200 column

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Hi-Load Superdex-200 column

It is very important to have fast and reasonably accurate technique to estimate the molecular weight of the protein globule. It is possible to use gel filtration results as a measurement of the protein molecular weight.

Please remember that gel filtration is not an accurate method to estimate MW or oligomeric state of the protein. Proteins are separated by their size and not by MW. Proteins with the same MW could have a different shape and compactness therefore their sizes could be different as well as elution volume (Ve) and apparent MW. Another possibility for misidentification is if the protein has some affinity to the matrix and elutes later than it should according to its MW.

In our calibrations we use Hi-Load Superdex 200 1.6x60cm columns from Amersham Bioscience:

Total volume: 120 ml
Void volume (Vo): 45ml
Separation range MW: 600KDa – 10KDa
Sample volume: 0.5-2ml, no restrictions on buffer composition
Loading: up to 50 mg of total protein
Flow rate: 1-1.5 ml/min
Elution: with continuous buffer

Ve/Vo vs protein MW calculator:

Ve/Vo: Calculate protein MW
MW kDa: Calculate Ve/Vo

Calibration plot for Hi-Load Superdex 200 column

This is a Calibration plot for Hi-Load Superdex 200 column.

Hi-Load Superdex 200 column calibration plot