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Strong affinity for monoclonal mouse IgG1 and rat IgG. Complete kit contains HiTrap Protein G HP (1 x 1 ml), accessories, pre-made buffers for 10 purifications and detailed experimental protocols. Protein G Sepharose 4 Fast Flow Supplied as a suspension ready for column packing. Human IgG, > 20 mg/ml medium 400 cm/h* Cow IgG, 23 mg/ml medium Goat IgG, 19 mg/ml medium Guinea pig IgG, 17 mg/ml medium Mouse IgG, 10 mg/ml medium Rat IgG, 7 mg/ml medium * See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate.
4. Wash with at least 10 column volumes of binding buffer or until no material appears in the eluent, as monitored at A280. 5. Elute the IgY with 10 column volumes of elution buffer. 6. Wash the column with 8 column volumes of wash buffer. 7. Immediately re-equilibrate the column with 5 column volumes of binding buffer. 8 M Na2SO4. The sample should have the same concentration of Na2SO4 as the binding buffer. An increase in salt concentration will reduce the purity of the eluted IgY. The purity of the eluted IgY may be improved by using gradient elution with, for example, a linear gradient 0–100% elution buffer over 10 column volumes, followed by elution with 100% elution buffer for a few column volumes.
4. Equilibrate the column with 10 column volumes of binding buffer. 5. Apply sample at a flow rate 1–4 ml/min (1 ml column) or 5 ml/min (5 ml column). Collect the flow-through fraction. A pump is more suitable for application of sample volumes greater than 15 ml. 6. Wash with 10 column volumes of binding buffer. Collect wash fraction. 7. Elute with 5 column volumes of elution buffer. Collect eluted fractions in small fractions such as 1 ml to avoid dilution of the eluate. 8. Wash with 10 column volumes of binding buffer.