IgG Purifications

IgG Purification

Note: Content of gel is 1 ml of gel in each column

1. Wash columns with 10 ml of 1 X PBS.
2. Add 2.5 ml of serum to 2.5 ml of PBS and divide this mixture into two IgG columns.
3. Collect the serum into a test tube as it drains through the column.
4. When serum finishes draining, pour the washed serum back into the column and begin collecting flow through again.  Repeat this step 5 to 6 times.
5. Wash the columns with 10 ml of 1 X PBS.
6. Prepare several 1 ml tubes with 50µl of 1M Tris (pH = 9.5).
7. Add 1 ml of Elution Buffer (100mM Glycine pH=2.8) to each tube and collect 1 ml of flow through.
8. Move to the next prepared tube and repeat step 7.
9. Test each 1 ml sample by preparing ELISA plate with 10µl of Bradford Assay and add 50µl of each 1 ml flow through.  Keep the samples that change the Bradford Assay from red to blue.
10. Dialyze the positive 1 ml samples together in 4 Liters of 1 X PBS at pH=7.2 for at least 24 hours.
11. Use spectrometer at 280 nm to find concentration of IgG in solution. (Extinction coefficient = 1.4)
12. To store IgG solution, keep frozen at -4^oC to -20^oC .