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Extra resources for Affinity Chromatography: Principles and Methods
The cleaved protein and thrombin wash through from the GSTrap FF column, thrombin binds to the HiTrap Benzamidine FF (high sub) column, and the eluted fractions contain pure cleaved protein. 55 Sample: 2 ml clarified E. 20 0 0 0 10 A) 15 A) 20 A) 25 B) 50 ml A) B) A) GSTrap FF, 1 ml B) HiTrap Benzamidine FF (high sub), 1 ml Mr 97 000 66 000 45 000 30 000 20 100 14 400 1 2 3 4 5 6 7 8 9 Gel: Lane Lane Lane Lane 1. 2. 3. 4. Lane Lane Lane Lane 5. 6. 7. 8. Lane 9. ExcelGel SDS Gradient 8–18%, Coomassie Blue staining Low Molecular Weight Calibration Kit (LMW) Clarified E.
Ready to use, prepacked columns, buffers and chemicals. High throughput when used with MicroPlex 24 Vacuum (up to 48 samples simultaneously). HisTrap Kit 12 mg*/column 4 ml/min As above, but includes buffers for up to 12 purifications using a syringe. HiTrap Chelating HP 1 ml 12 mg*/column 4 ml/min Prepacked column, ready to use. HiTrap Chelating HP 5 ml 60 mg*/column 20 ml/min Prepacked column, ready to use. Chelating Sepharose Fast Flow 12 mg*/ml medium 400 cm/h** Supplied as suspension for packing columns and scale up.
As an affinity ligand, protein A is coupled to Sepharose so that these regions are free to bind IgG. One molecule of protein A can bind at least two molecules of IgG. Both protein A and a recombinant protein A are available from Amersham Biosciences. These molecules share similar specificities for the Fc region of IgG, but the recombinant protein A has been engineered to include a C-terminal cysteine that enables a single-point coupling to Sepharose. Single point coupling often results in an enhanced binding capacity.