Calibration plot for Hi-Load Superdex-200 column
Main steps:
- Protein purification- Introduction
- Step-by-Step
- Common sense
- Protocols
- Charts & Tables
- AM concentration
- NaCl-sgar refractometer
- Calibration of Superdex 200
- AM table
- UV no TRP
- UV with TRP
- Appendix
- Crystallisation
Special:
- Programs for crystallography- X-ray detectors
Basic tutorials:
- Chemistry- Protein
- Peptide
- Amino Acids
Xtal community:
- CCP4BBProtocols and tips in protein purification or How to purify protein in one day.
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:
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Calibration plot for Hi-Load Superdex 200 column
This is a Calibration plot for Hi-Load Superdex 200 column.